Ahmed Mady

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.

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

Abstract 1173: A novel selective Mcl-1 inhibitor exhibitsin vitroandin vivoefficacy in melanoma

Metastatic melanoma is the deadliest form of skin cancer that still has limited treatment options and dismal 5-year year survival rates as low as 15%. Only recently has there been significant progress in the treatment of metastatic melanoma, advent by molecular targeted drugs and immunotherapy, but their limitations are exposed as these cancers are quickly able to develop resistance. Dysregulation of apoptotic machinery in melanoma allows the cancer cells to evade cell death and contributes to treatment resistance. Up-regulation of Mcl-1, a member of the Bcl-2 family of anti-apoptotic proteins, has been correlated with melanoma progression and metastasis. Mcl-1 amplification is one of the most common genetic aberrations found in human cancers and has been labeled as a marker of aggressive oncogenesis and poor patient prognosis. Previous studies have pointed to Mcl-1 as a viable therapeutic target for the treatment of melanoma and conclude that small molecule Mcl-1 inhibitors may appeal this unmet medical need. We have discovered and characterized a new class of selective small molecule Mcl-1 inhibitors using various biochemical, functional, and cell based assays; further development of these compounds allowed us to achieve potent low-nanomolar binding affinity for Mcl-1 and more than 300-fold selectivity over Bcl-2/Bcl-xL. Our most potent inhibitor, 483-LM, was screened across a panel of human melanoma cell lines using a cell proliferation assay and revealed varying levels of sensitivity. A BH3 profiling assay demonstrates that C8161, the cell line most sensitive to our inhibitor, solely relies on Mcl-1 for survival. The Mcl-1 dependence of C8161 might contribute to the known metastatic nature of this cell line. Mechanistic studies revealed that 483-LM effectively engaged the endogenous Mcl-1 protein after treatment of C8161, as determined by a CETSA assay, and prompted disruption of protein-protein interactions between Mcl-1 and several pro-apoptotic proteins, including Bax, Bak and Bim. This was followed by induction of Bax/Bak dependent apoptosis and activation of hallmarks of the intrinsic apoptotic pathway, including mitochondrial outer membrane depolarization, caspase activation, and PARP cleavage. Importantly, treatment with 483-LM caused massive up-regulation of the pro-apoptotic BH3-only protein Noxa, an effect apparent after an 8 hour treatment, which contributes to the induction of cell death. The in vivo efficacy studies with 483-LM showed significant effect on tumor growth and caspase-3 activation in tumor samples. Overall, our data indicate that Mcl-1 inhibitors are a promising treatment option for aggressive metastatic melanoma and warrant further preclinical investigation of 483-LM as a promising selective Mcl-1 inhibitor to be used as a single agent and in combination with chemotherapy and immunotherapy. Citation Format: Karson J. Kump, Lei Miao, Ahmed S. Mady, Katherine Lev, William Giblin, Mary E. Skinner, David B. Lombard, Zaneta Nikolovska-Coleska. A novel selective Mcl-1 inhibitor exhibits in vitro and in vivo efficacy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1173. doi:10.1158/1538-7445.AM2017-1173

Abstract 3658: Structure-guided de novo design of selective Mcl-1 Inhibitors: Synthesis, structural and biochemical characterization

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Myeloid cell leukemia-1 (Mcl-1) is a potent anti-apoptotic protein, member of the prosurvival Bcl-2 family. Amplification of the gene encoding the Mcl-1 protein is a common genetic aberration in human cancer. Overexpression of Mcl-1 is associated with high tumor grade, resistance to chemotherapy and poor prognosis in many types of cancers. Thus Mcl-1 is emerging as a critical survival factor in a broad range of human cancers and represents an attractive molecular target for development of a new class of cancer therapy. There is still need for developing BH3 mimetics that can efficiently and selectively target Mcl-1 protein. Employing de-novo structure-based drug-design, we have designed a new class of potent small-molecule Mcl-1 inhibitors based on the initial lead compound discovered by high-throughput screening. Analyzing the binding model of this compound with Mcl-1 through computational docking supported by HSQC NMR studies, and using the structural information, we have designed and optimized a class of small-molecule inhibitors of Mcl-1 using a 2-(phenylsulfonamido) benzoic acid as a scaffold to reproduce the key interactions with Mcl-1 such as the conserved hydrogen bond between the aspartate in pro-apoptotic proteins and Arg263 in Mcl-1. Several co-crystal structures of this class inhibitors in complex with Mcl-1 have provided a basis for their further optimization which ultimately led to the discovery of ligands with low-nanomolar potency (Ki = 8 nM) and selectivity for Mcl-1. These compounds bind in the canonical BH3 binding groove. Interestingly, depending on the particular substitution pattern in the core scaffold, a “flip’ binding mode was observed where the substituents bind in a reverse orientation. These findings were confirmed by X-ray crystallography. The most potent compounds were further biologically characterized with a series of complementary biochemical and functional assays. Citation Format: Ahmed Mady, Lei Miao, Andrej Perdih, Chenzhong Liao, Naval Bajwa, Jeanne Stuckey, Zaneta Nikolovska-Coleska. Structure-guided de novo design of selective Mcl-1 Inhibitors: Synthesis, structural and biochemical characterization. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3658. doi:10.1158/1538-7445.AM2015-3658

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