Tony Cheung

Structural Basis of Substrate Methylation and Inhibition of SMYD2.

Protein lysine methyltransferases are important regulators of epigenetic signaling. These enzymes catalyze the transfer of donor methyl groups from S-adenosylmethionine to specific acceptor lysines on histones, leading to changes in chromatin structure and transcriptional regulation. These enzymes also methylate nonhistone protein substrates, revealing an additional mechanism to regulate cellular physiology. The oncogenic protein SMYD2 represses the functional activities of the tumor suppressor proteins p53 and Rb, making it an attractive drug target. Here we report the discovery of AZ505, a potent and selective inhibitor of SMYD2 that was identified from a high throughput chemical screen. We also present the crystal structures of SMYD2 with p53 substrate and product peptides, and notably, in complex with AZ505. This substrate competitive inhibitor is bound in the peptide binding groove of SMYD2. These results have implications for the development of SMYD2 inhibitors, and indicate the potential for developing novel therapies targeting this target class.

Aplysamine 6, an Alkaloidal Inhibitor of Isoprenylcysteine Carboxyl Methyltransferase from the Sponge Pseudoceratina sp.

The anticancer target isoprenylcysteine carboxyl methyltransferase (Icmt) was the focus of a natural product high-throughput screening campaign. The Australian marine sponge Pseudoceratina sp. yielded aplysamine 6, a new bromotyrosine derivative with an alpha,beta-unsaturated amide linkage, as the bioactive constituent. Its structure was determined by 1D and 2D NMR spectroscopy.

Biochemical Characterization of Human SET and MYND Domain-Containing Protein 2 Methyltransferase

SET and MYND domain-containing protein 2 (SMYD2) is a protein lysine methyltransferase that catalyzes the transfer of methyl groups from S-adenosylmethionine (AdoMet) to acceptor lysine residues on histones and other proteins. To understand the kinetic mechanism and the function of individual domains, human SMYD2 was overexpressed, purified, and characterized. Substrate specificity and product analysis studies established SMYD2 as a monomethyltransferase that prefers nonmethylated p53 peptide substrate. Steady-state kinetic and product inhibition studies showed that SMYD2 operates via a rapid equilibrium random Bi Bi mechanism at a rate of 0.048 ± 0.001 s(-1), with K(M)s for AdoMet and the p53 peptide of 0.031 ± 0.01 μM and 0.68 ± 0.22 μM, respectively. Metal analyses revealed that SMYD2 contains three tightly bound zinc ions that are important for maintaining the structural integrity and catalytic activity of SMYD2. Catalytic activity was also shown to be dependent on the GxG motif in the S-sequence of the split SET domain, as a G18A/G20A double mutant and a sequence deletion within the conserved motif impaired AdoMet binding and significantly decreased enzymatic activity. The functional importance of other SMYD2 domains including the MYND domain, the cysteine-rich post-SET domain, and the C-terminal domain (CTD), were also investigated. Taken together, these results demonstrated the functional importance of distinct domains in the SMYD family of proteins and further advanced our understanding of the catalytic mechanism of this family.

AZD5153: a novel bivalent BET bromodomain inhibitor highly active against hematologic malignancies

The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Pharmacological targeting of BRD4 bromodomains by small molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs critical for tumor growth and/or survival. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor possessing a bivalent binding mode. Unlike previously described monovalent inhibitors, AZD5153 ligates two bromodomains in BRD4 simultaneously. The enhanced avidity afforded through bivalent binding translates into increased cellular and antitumor activity in preclinical hematologic tumor models. In vivo administration of AZD5153 led to tumor stasis or regression in multiple xenograft models of acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. The relationship between AZD5153 exposure and efficacy suggests that prolonged BRD4 target coverage is a primary efficacy driver. AZD5153 treatment markedly affects transcriptional programs of MYC, E2F, and mTOR. Of note, mTOR pathway modulation is associated with cell line sensitivity to AZD5153. Transcriptional modulation of MYC and HEXIM1 was confirmed in AZD5153-treated human whole blood, thus supporting their use as clinical pharmacodynamic biomarkers. This study establishes AZD5153 as a highly potent, orally available BET/BRD4 inhibitor and provides a rationale for clinical development in hematologic malignancies. Mol Cancer Ther; 15(11); 2563–74. ©2016 AACR.

Discovery and Optimization of a Novel Series of Potent Mutant B-Raf V600E Selective Kinase Inhibitors.

B-Raf represents an attractive target for anticancer therapy and the development of small molecule B-Raf inhibitors has delivered new therapies for metastatic melanoma patients. We have discovered a novel class of small molecules that inhibit mutant B-Raf(V600E) kinase activity both in vitro and in vivo. Investigations into the structure-activity relationships of the series are presented along with efforts to improve upon the cellular potency, solubility, and pharmacokinetic profile. Compounds selectively inhibited B-Raf(V600E) in vitro and showed preferential antiproliferative activity in mutant B-Raf(V600E) cell lines and exhibited selectivity in a kinase panel against other kinases. Examples from this series inhibit growth of a B-Raf(V600E) A375 xenograft in vivo at a well-tolerated dose. In addition, aminoquinazolines described herein were shown to display pERK elevation in nonmutant B-Raf cell lines in vitro.

Discovery of pyrazolo[1,5-a]pyrimidine B-cell lymphoma 6 (BCL6) binders and optimization to high affinity macrocyclic inhibitors

Inhibition of the protein-protein interaction between B-cell lymphoma 6 (BCL6) and corepressors has been implicated as a therapeutic target in diffuse large B-cell lymphoma (DLBCL) cancers and profiling of potent and selective BCL6 inhibitors are critical to test this hypothesis. We identified a pyrazolo[1,5-a]pyrimidine series of BCL6 binders from a fragment screen in parallel with a virtual screen. Using structure-based drug design, binding affinity was increased 100000-fold. This involved displacing crystallographic water, forming new ligand-protein interactions and a macrocyclization to favor the bioactive conformation of the ligands. Optimization for slow off-rate constant kinetics was conducted as well as improving selectivity against an off-target kinase, CK2. Potency in a cellular BCL6 assay was further optimized to afford highly selective probe molecules. Only weak antiproliferative effects were observed across a number of DLBCL lines and a multiple myeloma cell line without a clear relationship to BCL6 potency. As a result, we conclude that the BCL6 hypothesis in DLBCL cancer remains unproven.

AZ304, a novel dual BRAF inhibitor, exerts anti-tumour effects in colorectal cancer independently of BRAF genetic status

BackgroundBRAF mutation is associated with poor clinical outcome of patients with malignant tumours, and mediates resistance to chemotherapy and targeted therapy. This study aimed to determine whether V600E mutant and wild type BRAF colorectal cancers exhibit distinct sensitivities to the dual BRAF inhibitor AZ304.MethodsKinase activity was assessed by the AlphaScreen assay. Then, MTT assay, EdU assay, colony-formation assay and Western blot were performed to evaluate the anti-tumour effects of AZ304 in vitro. In vivo efficacy was investigated by xenograft analysis and immunohistochemistry.ResultsAZ304 exerted potent inhibitory effects on both wild type and V600E mutant forms of the serine/threonine-protein kinase BRAF, with IC50 values of 79 nM and 38 nM, respectively. By suppressing ERK phosphorylation, AZ304 effectively inhibited a panel of human cancer cell lines with different BRAF and RAS genetic statuses. In selected colorectal cancer cell lines, AZ304 significantly inhibited cell growth in vitro and in vivo, regardless of BRAF genetic status. In addition, the EGFR inhibitor Cetuximab enhanced the potency of AZ304 independently of BRAF mutational status.ConclusionsThe BRAF inhibitor AZ304 has broad spectrum antitumour activity, which is significantly enhanced by combination with Cetuximab in colorectal cancers in vitro and in vivo.

Development of a Novel B-Cell Lymphoma 6 (BCL6) PROTAC To Provide Insight into Small Molecule Targeting of BCL6

B-cell lymphoma 6 (BCL6) inhibition is a promising mechanism for treating hematological cancers but high quality chemical probes are necessary to evaluate its therapeutic potential. Here we report potent BCL6 inhibitors that demonstrate cellular target engagement and exhibit exquisite selectivity for BCL6 based on mass spectrometry analyses following chemical proteomic pull down. Importantly, a proteolysis-targeting chimera (PROTAC) was also developed and shown to significantly degrade BCL6 in a number of diffuse large B-cell lymphoma (DLBCL) cell lines, but neither BCL6 inhibition nor degradation selectively induced marked phenotypic response. To investigate, we monitored PROTAC directed BCL6 degradation in DLBCL OCI-Ly1 cells by immunofluorescence and discovered a residual BCL6 population. Analysis of subcellular fractions also showed incomplete BCL6 degradation in all fractions despite having measurable PROTAC concentrations, together providing a rationale for the weak antiproliferative response seen with both BCL6 inhibitor and degrader. In summary, we have developed potent and selective BCL6 inhibitors and a BCL6 PROTAC that effectively degraded BCL6, but both modalities failed to induce a significant phenotypic response in DLBCL despite achieving cellular concentrations.

Abstract 4705: Therapeutic activity of bivalent BRD4 inhibitor AZD5153 in hematological cancers

The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Across a number of tumor models pharmacological targeting of BRD4 bromodomains by small-molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor. AZD5153 is a candidate drug that possesses an unprecedented bivalent binding mode among reported BET inhibitors, which allows AZD5153 to ligate the tandem bromodomains in BRD4. The avidity resulted from the bivalent binding interaction translates into markedly enhanced cellular potency. Although AZD5153 demonstrates broad activity across a cancer cell line panel comprising solid and hematologic subtypes, there is enriched antitumor activity against hematologic cell lines, including acute myeloid leukemia (AML), multiple myeloma (MM), and diffuse large B-cell lymphoma (DLBCL). The activity of AZD5153 in hematologic tumors was further confirmed in five selected xenograft models of AML, MM, and DLBCL where AZD5153 treatment led to tumor stasis or regression, accompanied by concomitant modulation of BRD4 pharmacodynamic markers, such as MYC and HEXIM1. In order to characterize the transcriptional consequences elicited by AZD5153, we carried out transcriptional profiling of 11 hematologic tumor lines and identified the robust modulation of MYC, and E2F transcriptional programs. Moreover, our transcriptional data was used to identify candidate clinical PD biomarkers. The suitability and dynamic range of the top two candidate biomarkers for AZD5153 was confirmed using human whole blood from normal healthy volunteers. To identify protein biomarkers associated with sensitivity to AZD5153 treatment, we deployed reverse-phase protein array (RPPA) technology to quantitatively examine the level of 182 proteins following AZD5153 treatment. Our findings indicate that cell lines sensitive to AZD5153 uniquely exhibit a marked decrease in the level of mTOR-pathway associated proteins following AZD5153 treatment. Conversely, MYC modulation was observed in both sensitive and resistant groups. Thus, these data suggest that in hematologic malignancies, mTOR pathway downregulation may serve as an appropriate biomarker of sensitivity to BRD4 inhibitors such as AZD5153. Our study establishes AZD5153 as a novel and potent BRD4/BET inhibitor possessing a unique bivalent binding property. We have characterized the pharmacological consequences of BRD4/BET inhibition by AZD5153 via unbiased transcriptional and proteome profiling. These efforts are the first to identify mTOR modulation as a putative biomarker of sensitivity to BET bromodomain inhibition in hematologic tumors and may help to inform future clinical evaluation of AZD5153 and other BET bromodomain inhibitors. Citation Format: Huawei (Ray) Chen, Maureen Hattersley, Garrett Rhyasen, Austin Dulak, Wendy Wang, Phil Petteruti, Ian Dale, Tony Cheung, Shenghua Wen, Lilian Castriotta, Deborah Lawson, Mike Collins, Miika Ahdesmaki, Graeme Walker, Al Rabow, Jonathan Dry, Corinne Reimer, Paul Lyne, Steve Fawell, MIke Waring, Mike Zinda, Ed Clark, Ed Clark. Therapeutic activity of bivalent BRD4 inhibitor AZD5153 in hematological cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4705.