Fardokht A. Abulwerdi

A Novel Small-Molecule Inhibitor of Mcl-1 Blocks Pancreatic Cancer Growth In Vitro and In Vivo

Using a high-throughput screening (HTS) approach, we have identified and validated several small-molecule Mcl-1 inhibitors (SMI). Here, we describe a novel selective Mcl-1 SMI inhibitor, 2 (UMI-77), developed by structure-based chemical modifications of the lead compound 1 (UMI-59). We have characterized the binding of UMI-77 to Mcl-1 by using complementary biochemical, biophysical, and computational methods and determined its antitumor activity against a panel of pancreatic cancer cells and an in vivo xenograft model. UMI-77 binds to the BH3-binding groove of Mcl-1 with Ki of 490 nmol/L, showing selectivity over other members of the antiapoptotic Bcl-2 family. UMI-77 inhibits cell growth and induces apoptosis in pancreatic cancer cells in a time- and dose-dependent manner, accompanied by cytochrome c release and caspase-3 activation. Coimmunoprecipitation experiments revealed that UMI-77 blocks the heterodimerization of Mcl-1/Bax and Mcl-1/Bak in cells, thus antagonizing the Mcl-1 function. The Bax/Bak-dependent induction of apoptosis was further confirmed using murine embryonic fibroblasts that are Bax- and Bak-deficient. In an in vivo BxPC-3 xenograft model, UMI-77 effectively inhibited tumor growth. Western blot analysis in tumor remnants revealed enhancement of proapoptotic markers and significant decrease of survivin. Collectively, these promising findings show the therapeutic potential of Mcl-1 inhibitors against pancreatic cancer and warrant further preclinical investigations. Mol Cancer Ther; 13(3); 565–75. ©2013 AACR.

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 Mcl-1 for Radiosensitization of Pancreatic Cancers

In order to identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer, we previously conducted an RNAi library screen of 8,800 genes. We identified Mcl-1 (myeloid cell leukemia-1), an anti-apoptotic member of the Bcl-2 family, as a target for sensitizing pancreatic cancer cells to chemoradiation. In the present study we investigated Mcl-1 inhibition by either genetic or pharmacological approaches as a radiosensitizing strategy in pancreatic cancer cells. Mcl-1 depletion by siRNA produced significant radiosensitization in BxPC-3 and Panc-1 cells in association with Caspase-3 activation and PARP cleavage, but only minimal radiosensitization in MiaPaCa-2 cells. We next tested the ability of the recently identified, selective, small molecule inhibitor of Mcl-1, UMI77, to radiosensitize in pancreatic cancer cells. UMI77 caused dissociation of Mcl-1 from the pro-apoptotic protein Bak and produced significant radiosensitization in BxPC-3 and Panc-1 cells, but minimal radiosensitization in MiaPaCa-2 cells. Radiosensitization by UMI77 was associated with Caspase-3 activation and PARP cleavage. Importantly, UMI77 did not radiosensitize normal small intestinal cells. In contrast, ABT-737, an established inhibitor of Bcl-2, Bcl-XL, and Bcl-w, failed to radiosensitize pancreatic cancer cells suggesting the unique importance of Mcl-1 relative to other Bcl-2 family members to radiation survival in pancreatic cancer cells. Taken together, these results validate Mcl-1 as a target for radiosensitization of pancreatic cancer cells and demonstrate the ability of small molecules which bind the canonical BH3 groove of Mcl-1, causing displacement of Mcl-1 from Bak, to selectively radiosensitize pancreatic cancer cells.

BH3 Profiling Reveals Selectivity by Herpesviruses for Specific Bcl-2 Proteins To Mediate Survival of Latently Infected Cells

ABSTRACT Herpesviruses, including human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and Kaposi’s sarcoma-associated herpesvirus, establish latency by modulating or mimicking antiapoptotic Bcl-2 proteins to promote survival of carrier cells. BH3 profiling, which assesses the contribution of Bcl-2 proteins towards cellular survival, was able to globally determine the level of dependence on individual cellular and viral Bcl-2 proteins within latently infected cells. Moreover, BH3 profiling predicted the sensitivity of infected cells to small-molecule inhibitors of Bcl-2 proteins.

Abstract 2917: Design, synthesis and biological evaluation of two chemical classes as novel small molecule Mcl-1 inhibitors

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The anti-apoptotic myeloid cell leukemia protein Mcl-1, a member of the Bcl-2 family proteins, has a critical and distinct role(s) in maintaining cell survival and is emerging as an independent and promising therapeutic target. Using structure-based design, two classes of small molecule Mcl-1 inhibitors were developed with IC50 values ranging from 50 nM to 75 μM, based on the lead compounds identified from high throughput screening. The structure-activity relationships (SAR) of these compounds were analyzed and chemical features of both series that are important for potency and selectivity on Mcl-1 were identified. The HSQC-NMR spectroscopy studies combined with molecular docking and dynamics simulations demonstrated that both classes of compounds occupy the BH3 binding site in Mcl-1 protein mimicking two conserved hydrophobic residues from BH3 binding motif. The most potent analogues antagonize Mcl-1 on the functional level and they induce release of cytochrome c, inhibit cell growth and induce apoptosis in Bax/Bak-dependent manner in pancreatic and melanoma cancer cells with high levels of Mcl-1. The knowledge gained through these studies provides promise for future design and expansion of these series towards development of potent small-molecule Mcl-1 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2917. doi:1538-7445.AM2012-2917

Mcl-1 small-molecule inhibitors encapsulated into nanoparticles exhibit increased killing efficacy towards HCMV-infected monocytes

Abstract Human cytomegalovirus (HCMV) spreads and establishes a persistent infection within a host by stimulating the survival of carrier myeloid cells via the upregulation of Mcl‐1, an antiapoptotic member of the Bcl‐2 family of proteins. However, the lack of potent Mcl‐1‐specific inhibitors and a targetable delivery system has limited the ability to exploit Mcl‐1 as a therapeutic strategy to eliminate HCMV‐infected monocytes. In this study, we found a lead compound from a novel class of Mcl‐1 small‐molecule inhibitors rapidly induced death of HCMV‐infected monocytes. Moreover, encapsulation of Mcl‐1 antagonists into myeloid cell‐targeting nanoparticles was able to selectively increase the delivery of inhibitors into HCMV‐activated monocytes, thereby amplifying their potency. Our study demonstrates the potential use of nanotechnology to target Mcl‐1 small‐molecule inhibitors to HCMV‐infected monocytes. HighlightsMcl‐1 inhibitors stimulate death of HCMV‐infected monocytes.HCMV‐infected monocytes exhibit enhanced uptake of nanoparticles.Encapsulated Mcl‐1 inhibitors induce more potent killing of infected monocytes.

Abstract 1641: Structure-based design and development of pyrazolopyridine-based inhibitors of Mcl-1

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The anti-apoptotic myeloid cell leukemia protein Mcl-1, a member of the Bcl-2 family proteins, has emerged as a promising therapeutic target. Mcl-1 is overexpressed in many human cancers which has been associated with inferior survival, poor prognosis and resistance to chemotherapies. Disruption of Mcl-1 interaction with its pro-apoptotic partners through small molecules is a viable strategy to overcome Mcl-1 mediated resistance to apoptosis. Targeting Mcl-1 protein represents a promising strategy either alone or in combination with other therapies. Applying an integrated screening approach through combining high throughput and virtual screenings, several novel chemical classes were identified as Mcl-1 inhibitors. Compound 38 with a pyrazolopyridine scaffold was selected as a promising high throughput lead for medicinal chemistry efforts. Using reported chemistry a focused library of analogs of 38 was generated. Through HSQC protein-observed NMR studies and chemical shift mapping, the binding of the lead compound 38 was characterized and confirmed to be the BH3 binding pocket of Mcl-1. Computational modeling guided by NMR studies was applied in lead optimization and rational design of more potent analogs. Structure-activity relationship was established utilizing two different competitive platforms of fluorescent polarization and surface plasmon resonance, and confirmed by HSQC NMR spectroscopy. The binding affinity of this class of compounds was improved more than twenty fold in comparison with the lead compound 38. In vitro binding, functional and cell-based assays were performed in order to determine selectivity profile against five members of Bcl-2 family, mechanism of action and cellular activity of analogs with improved potency. The obtained results show promise for further chemical modification and development of pyrazolopyridine-based inhibitors of Mcl-1. Citation Format: Fardokht A. Abulwerdi, Ahmed S.A. Mady, Andrej Perdih, Jeanne A. Stuckey, Hollis D. Showalter, Zaneta Nikolovska-Coleska. Structure-based design and development of pyrazolopyridine-based inhibitors of Mcl-1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1641. doi:10.1158/1538-7445.AM2014-1641

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 2822: Targeting Mcl-1 regulated apoptosis pathway as a novel therapy in pancreatic cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Resistance of pancreatic cancer to current treatment protocols as a consequence of apoptosis defects is a major cause of treatment failure. Therapeutic approaches that specifically target components of these molecular mechanisms are urgently needed. The anti-apoptotic protein Mcl-1, a member of the Bcl-2 family proteins, has emerged as a promising therapeutic target. Consistent with its anti-apoptotic function, the overexpression of Mcl-1 in pancreatic cancer cells has been associated with tumor progression and resistance to current chemotherapeutics. Applying high throughput screening approach several promising lead compounds as selective Mcl-1 inhibitors were identified and evaluated. Based on one of the most promising lead compounds we have designed and synthesized several analogues. The most potent compound, UMI-77, binds to Mcl-1 protein with Ki = 205 nM determined by fluorescence polarization based assay and shows selectivity over Bcl-2 and Bcl-xL. NMR spectroscopy studies demonstrate that UMI-77 binds to the same BH3 domain of Mcl-1 as the Bim BH3 peptide and antagonizes Mcl-1 function. UMI-77 inhibits cell growth and induces apoptosis in pancreatic cancer cells with high Mcl-1 levels (BxPC-3 and Panc-1) in a time and dose-dependent manner, accompanied by caspase-3 activation. Applying siRNA approach, the transient suppression of Mcl-1 abrogated UMI-77 mediated apoptosis in BxPC-3 cells, demonstrating that UMI-77 as a single agent can inhibit cell growth and induce apoptosis in pancreatic cells in a Mcl-1 dependent manner. Co-immunoprecipitation experiments revealed that UMI-77 blocks the heterodimerization of Mcl-1/Bax and Mcl-1/Bak in cells. 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 UMI-77 mainly depend on Bax and/or Bak, suggesting that function as BH3 mimetic. In order to verify the clinical utility of UMI-77, we tested its in vivo efficacy using BxPC-3 xenograft model. The maximum tolerated dose (MTD) of UMI-77 in SCID mice was determined to be 60mg/kg i.v. We further demonstrated that UMI-77 at MTD showed statistically significant and effective tumor growth inhibition in BxPC-3 xenograft model. At the end of the treatment, western blot analysis for different molecular markers on lysates isolated from tumors harvested from SCID mice revealed enhancement of proapoptotic markers in UMI-77 treated animals compared to control. Collectively, these promising findings warrant further chemical modifications of this compound and optimization toward developing a new class of anticancer drugs, Mcl-1 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2822. doi:10.1158/1538-7445.AM2011-2822