Chenxi Shen

3-Substituted-N-(4-hydroxynaphthalen-1-yl)arylsulfonamides as a novel class of selective Mcl-1 inhibitors: structure-based design, synthesis, SAR, and biological evaluation.

Mcl-1, an antiapoptotic member of the Bcl-2 family of proteins, is a validated and attractive target for cancer therapy. Overexpression of Mcl-1 in many cancers results in disease progression and resistance to current chemotherapeutics. Utilizing high-throughput screening, compound 1 was identified as a selective Mcl-1 inhibitor and its binding to the BH3 binding groove of Mcl-1 was confirmed by several different, but complementary, biochemical and biophysical assays. Guided by structure-based drug design and supported by NMR experiments, comprehensive SAR studies were undertaken and a potent and selective inhibitor, compound 21, was designed which binds to Mcl-1 with a Ki of 180 nM. Biological characterization of 21 showed that it disrupts the interaction of endogenous Mcl-1 and biotinylated Noxa-BH3 peptide, causes cell death through a Bak/Bax-dependent mechanism, and selectively sensitizes Eμ-myc lymphomas overexpressing Mcl-1, but not Eμ-myc lymphoma cells overexpressing Bcl-2. Treatment of human leukemic cell lines with compound 21 resulted in cell death through activation of caspase-3 and induction of apoptosis.

Targeting Recruitment of Disruptor of Telomeric Silencing 1-like (DOT1L) CHARACTERIZING THE INTERACTIONS BETWEEN DOT1L AND MIXED LINEAGE LEUKEMIA (MLL) FUSION PROTEINS

Background: MLL fusion proteins use similar strategy for leukemic transformation through DOT1L recruitment. Results: Ten amino acids in DOT1L were identified as essential for binding and transformation by MLL-AF9. Conclusion: Biochemical and functional results indicate that blocking DOT1L recruitment represents a promising therapeutic strategy for mixed lineage leukemia. Significance: Identified DOT1L peptide will lay a foundation toward discovery of chemical tools able to block DOT1L recruitment. The MLL fusion proteins, AF9 and ENL, activate target genes in part via recruitment of the histone methyltransferase DOT1L (disruptor of telomeric silencing 1-like). Here we report biochemical, biophysical, and functional characterization of the interaction between DOT1L and MLL fusion proteins, AF9/ENL. The AF9/ENL-binding site in human DOT1L was mapped, and the interaction site was identified to a 10-amino acid region (DOT1L865–874). This region is highly conserved in DOT1L from a variety of species. Alanine scanning mutagenesis analysis shows that four conserved hydrophobic residues from the identified binding motif are essential for the interactions with AF9/ENL. Binding studies demonstrate that the entire intact C-terminal domain of AF9/ENL is required for optimal interaction with DOT1L. Functional studies show that the mapped AF9/ENL interacting site is essential for immortalization by MLL-AF9, indicating that DOT1L interaction with MLL-AF9 and its recruitment are required for transformation by MLL-AF9. These results strongly suggest that disruption of interaction between DOT1L and AF9/ENL is a promising therapeutic strategy with potentially fewer adverse effects than enzymatic inhibition of DOT1L for MLL fusion protein-associated leukemia.

In silico discovery of acylated flavonol monorhamnosides from Eriobotrya japonica as natural, small-molecular weight inhibitors of XIAP BIR3

Targeting the baculoviral inhibitor of apoptosis proteins repeat (BIR) 3 of X-linked inhibitor of apoptosis proteins (XIAP) represents an innovative strategy for the design of chemosensitizers. Acylated flavonol monorhamnosides (AFMR) from Eriobotrya japonica Lindl. (Rosaceae) were virtually predicted as ligands of the XIAP BIR3 domain by using a previously generated pharmacophore model. From the methanol leaf extract of E. japonica an enriched mixture of AFMR was obtained showing chemosensitizing potential in combination with etoposide in XIAP-overexpressing Jurkat cells. The HPLC-SPE-NMR hyphenated technique facilitated the structure elucidation of three known and two new natural AFMR. The main constituent and virtual hit, kaempferol-3-O-α-l-(2″,4″-di-E-p-coumaroyl)-rhamnoside (3) was isolated from the enriched fraction. Applying a fluorescence polarization based binding assay, 3 was identified as XIAP BIR3 ligand with a dose-dependent affinity (IC₅₀ 10.4 μM). Further, 3 induced apoptosis in XIAP-overexpressing Jurkat cells and activated caspase-9 in combination with etoposide. Docking experiments revealed a major impact of the coumaric acid and sugar moieties of 3 on XIAP BIR3 binding, which was experimentally confirmed. To conclude, this study elucidates 3 as natural, small-molecular weight XIAP BIR3 inhibitor using a combination of in silico and HPLC-SPE-NMR hyphenated techniques.

Abstract 3803: Development of genetic and chemical tools for understanding the recruitment of DOT1L in MLL-fusion driven leukemia and normal hematopoiesis

Leukemias harboring rearrangements of mixed-lineage leukemia gene (MLL1) are associated with poor clinical outcomes, and new therapeutic approaches are needed. Rearrangements of the MLL1 gene generate fusion oncoproteins which drive the high expression of the clustered homeobox (HOX) genes and induce leukemic transformation. Genome wide histone methylation studies have revealed that the abnormal expression of MLL1 fusion target genes is associated with high levels of histone H3 lysine 79 (H3K79) methylation. Recruitment of DOT1L (disruptor of telomeric silencing 1-like), a unique histone methyltransferase that catalyzes methylation of H3K79, proved to be essential for the transforming activity of multiple MLL fusion proteins. To gain insights into the unique functions of DOT1L in MLL-driven leukemia, we elucidated the mechanisms of DOT1L recruitment to the MLL fusion partners. The binding site was mapped to a short segment of 10 amino acids in DOT1L and peptides derived from this region disrupted the interaction between DOT1L and MLL-AF9. DOT1L mutants lacking these 10 residues did not support transformation by MLL-AF9. This discovery has established a foundation for disease-specific therapies that target chromatin modifications in highly malignant leukemias. Applying high throughput screening approach several different chemical classes of small molecules that disrupt the protein-protein interactions between DOT1L and oncogenic MLL-fusion proteins were identified and validated. To evaluate if the AF9-binding domain of DOT1L is critical for its functions in normal hematopoietic stem cells as opposed to leukemias driven by MLL fusion proteins, genetic tools were developed to functionally investigate the importance of the DOT1L AF9-binding domain in MLL-AF9-driven leukemia and its role in the physiological functions of DOT1L in normal hematopoiesis. Our findings demonstrate that pharmacological inhibition of the DOT1L complex through disrupting the AF9-DOT1L interactions may provide therapeutic benefits in an array of malignancies with abnormal HOXA gene expression. Citation Format: Sierrah Grigsby, Jennifer Chase, James Ropa, Justin Serio, Chenxi Shen, Martha Larsen, Preston Donover, Melvin Reichman, Andrew Muntean, Ivan Maillard, Zaneta Nikolovska-Coleska. Development of genetic and chemical tools for understanding the recruitment of DOT1L in MLL-fusion driven leukemia and normal hematopoiesis. [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 3803.

Abstract 2469: Identification of novel Mcl-1 inhibitors using integrated screening approach: combining high throughput and virtual screening.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Myeloid cell leukemia-1 (Mcl-1) is a potent anti-apoptotic protein, a member of the prosurvival Bcl-2 family, and its role is emerging as a critical survival factor in a broad range of human cancers. Mcl-1 represents a very attractive molecular target for development of a new class of cancer therapy and there is still need for developing BH3 mimetics that can efficiently and selectively target Mcl-1 protein. We employed integrated screening approach through combining high throughput and virtual screening, to identify novel chemical classes as Mcl-1 inhibitors. For this purpose we developed a dual-readout HTS assay that combines two assay technologies, FP and FRET, into one system using the Mcl-1 and labeled Noxa BH3 derived peptide and optimized in a 1,536-well ultra-HTS format. This assay was used for screening a library of 102,255 compounds. As two assay platforms were utilized for the same target simultaneously, hit information was enriched identifying 1214 compounds. To further improve the output and the quality of the identified inhibitors, as well as to incorporate the structure-based knowledge of the interactions between Mcl-1 and number of BH3 peptides, we have integrated in silico target-based screening for selection of the most promising hits for further validation. The complex structure between Mcl-1 and Noxa BH3 peptide (PDB ID: [2NLA][1]) was used for the induced fit docking of 1214 hits. Followed by the scoring and ranking of the identified hits, 62 compounds were selected for further evaluation in a series of complementary biochemical, biophysical and functional assays. Several in vitro binding assays with different platform, FP, SPR, and (HSQC) NMR spectroscopy, were used to determine the binding affinity of the compounds. The binding data revealed that we have identified inhibitors with different selectivity profiles against five members of the Bcl-2 family: Mcl-1, Bcl-2, Bcl-xL, Bcl-w and A1. 15N HSQC spectra conclusively showed that newly identified compounds interact with the BH3 domain in Mcl-1 protein and affect many residues in the BH3 binding groove. Using pull down assay it was demonstrated that the identified compounds were able in a dose dependent manner to disrupt interactions between endogenous Mcl-1 protein and biotin labeled Noxa BH3 peptide. Functional studies showed that compounds can antagonize Mcl-1 function and induce cytochrome c and Smac release from the isolated mitochondria. By using murine embryonic fibroblasts (MEFs), wild type and deficient in both Bax and Bak (double knock out), it was further demonstrated that the cytotoxic activity and induction of apoptosis, depend on Bax and/or Bak, suggesting that the compounds function as BH3 mimetics. Collectively, these findings provide several different chemical classes for further chemical modifications and optimization toward developing a new class of anticancer drugs as Mcl-1 inhibitors. Citation Format: Ahmed Mady, Chenzhong Liao, Fardokht Abulwerdi, Chenxi Shen, Yuhong Du, Jeanne Stuckey, Haian Fu, Zaneta Nikolovska-Coleska. Identification of novel Mcl-1 inhibitors using integrated screening approach: combining high throughput and virtual screening. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2469. doi:10.1158/1538-7445.AM2013-2469 [1]: /lookup/external-ref?link_type=PDB&access_num=2NLA&atom=%2Fcanres%2F73%2F8_Supplement%2F2469.atom

Abstract A158: Therapeutic strategies for targeting histone methyltransferase DOT1L in MLL rearranged leukemia.

MLL fusion proteins activate target genes in part via recruitment of the histone-modifying enzyme, DOT1L (disruptor of telomeric silencing 1-like), a unique histone methyltransferase with selective activity for histone H3 lysine 79 (H3K79). The resulting hypermethylation of H3K79 at the HOX and MEIS1 loci appears to be pivotal for leukemogenesis in both AML and ALL. These findings emphasize the central role of DOT1L in leukemogenesis and strongly suggest that pharmacologic inhibition of DOT1L could be of therapeutic benefit for a number of ALL and AML subtypes. Applying a biochemical screening approach, two classes of small molecule DOT1L substrate competitive inhibitors were discovered. They selectively inhibit the cell growth in leukemic cells with MLL-translocation, in comparison with leukemic cells without MLL translocation, resulting in a dose and time dependent decrease of cellular H3K79 methylation, decrease in expression of MLL-target genes HOXA9 and MEIS1, inducing G0/G1 cell cycle arrest, an increase in expression of differentiation markers and death by apoptosis. Although the DOT1L inhibitors showed no toxicity on normal bone marrow, knockout studies show that DOT1L deficiency is associated with chromosomal instability and fatal progressive anemia. This suggested that developing therapeutic strategies other than global inhibition of DOT1L activity may be necessary. Therefore we characterized the protein-protein interaction (PPI) between DOT1L and ENL/AF9 and assessed its importance on MLL fusion protein-mediated transformation. We found that ENL and AF9 C-terminal domains bind to DOT1L with Kd of 238 nM and 111 nM respectively. We have mapped the binding site in DOT1L and found that only 10 amino acid of DOT1L are involved in interaction with ENL and AF9. Synthetic DOT1L 10mer peptide binds ENL/AF9 and disrupts the interaction between DOT1L/ENL and DOT1L/AF9. Alanine scanning mutagenesis studies showed that four hydrophobic residues are detrimental for the binding of DOT1L to both proteins ENL/AF9. To examine the critical role of the DOT1L-AF9 interaction in transformation by MLL fusion protein, colony forming unit (CFU) assay was performed using MLL-AF9 transformed cells lacking endogenous DOT1L. The colony forming potential of the MLL-immortalized cells was completely abolished by introducing DOT1L construct lacking 10 amino acid AF9 interacting residues (DOT1L (Δ10aa)), while the wild type DOT1L construct was able to restore CFU formation. Importantly global levels of H3K79 methylation were equivalent, but only the DOT1L (Δ10aa) construct failed to restore CFU formation. These results demonstrate that DOT1L interaction with MLL-AF9 in addition to H3K79 methylation is required for transformation of MLL-AF9. This suggests that selective disruption of this PPI is a promising therapeutic strategy with potentially fewer adverse effects than enzymatic inhibition of DOT1L for MLL-fusion protein associated leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A158.

Abstract 4521: Discovery of novel small molecule inhibitors of the antiapoptotic protein Mcl-1 through high-throughput screening approach

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Myeloid cell leukemia (Mcl-1) is a multidomain, anti-apoptotic protein, member of the Bcl-2 family proteins, an important survival factor for many cancers. Mcl-1 has a critical and distinct role(s) in maintaining cell survival and emerging as an independent and promising therapeutic target. Through high throughout screening (HTS) of 53,300 structurally diverse synthetic organic compounds, we have discovered a number of novel inhibitors of Mcl-1. The hits identified by HTS were evaluated in a series of complementary biochemical, biophysical, functional and cellular assays to eliminate false positives and to determine the specificity and mechanism of action of these new compounds and 23 compounds were found to be inhibitors of Mcl-1. The most promising compounds, 59 and 62, have diverse chemical scaffolds. We have shown that these small molecule inhibitors bind to the BH3 binding site in Mcl-1 with IC50 values of 1,200 nM and 200 nM, respectively, and compete with BH3 peptides derived from Bid, Bim, or Noxa proteins. They bind to the Mcl-1 protein showing selectivity over two other Bcl-2 family members, Bcl-2 and Bcl-xL. NMR spectroscopy shows that 59 and 62 bind to the same BH3 domain of Mcl-1 as the Bim BH3 peptide. These initial lead compounds antagonize Mcl-1 on the functional level and they induce release of cytochrome c, inhibit cell growth and induce apoptosis in pancreatic and melanoma cancer cells with high levels of Mcl-1. We have tested several analogues of 59 and 62, both synthetic and commercially available, and established initial structure-activity relationships. One of the compounds tested (77), an analogue of 59, has improved binding affinity to Mcl-1 protein, as well as potent activity in inhibition of cell growth in cancer cells. Furthermore, by using murine embryonic fibroblasts (MEFs), wild type and deficient in both Bax and Bak (double knock out), it was demonstrated that the cytotoxic activity and induction of apoptosis by several analogues of the lead compound 59, depend on Bax and/or Bak, suggesting that they function as BH3 mimetics. Collectively, these findings provide good promise for chemical modifications of the new identified lead compounds, 59 and 62, and their further optimization toward developing a new class of anticancer drugs, Mcl-1 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4521.