Andrew M. Lewis

A thalidomide analogue with in vitro antiproliferative, antimitotic, and microtubule-stabilizing activities

We discovered a thalidomide analogue [5-hydroxy-(2,6-diisopropylphenyl)-1H-isoindole-1,3-dione (5HPP-33)] with antiproliferative activity against nine cancer cell lines in vitro. Flow cytometric analyses showed that the compound caused G2-M arrest, which occurred mainly at the mitotic phase. In addition, immunofluorescence microscopy and in vitro tubulin polymerization studies showed that 5HPP-33 has antimicrotubule activity with a paclitaxel-like mode of action. It is effective against four different paclitaxel-resistant cell lines. Thus, 5HPP-33 represents a potential antitumor agent. [Mol Cancer Ther 2006;5(2):450–6]

Inhibiting Aberrant Signal Transducer and Activator of Transcription Protein Activation with Tetrapodal, Small Molecule Src Homology 2 Domain Binders: Promising Agents against Multiple Myeloma

The signal transducer and activator of transcription (STAT) proteins represent a family of cytoplasmic transcription factors that regulate a pleiotropic range of biological processes. In particular, Stat3 protein has attracted attention as it regulates the expression of genes involved in a variety of malignant processes, including proliferation, survival, migration, and drug resistance. Multiple myeloma (MM) is an incurable hematologic malignancy that often exhibits abnormally high levels of Stat3 activity. Although current treatment strategies can improve the clinical management of MM, it remains uniformly incurable with a dismal median survival time post-treatment of 3-4 years. Thus, novel targeted therapeutics are critically needed to improve MM patient outcomes. We herein report the development of a series of small molecule Stat3 inhibitors with potent anti-MM activity in vitro. These compounds showed high-affinity binding to Stat3s SH2 domain, inhibited intracellular Stat3 phosphorylation, and induced apoptosis in MM cell lines at low micromolar concentrations.

STAT3 pathway regulates lung-derived brain metastasis initiating cell capacity through miR-21 activation.

Brain metastases (BM) represent the most common tumor to affect the adult central nervous system. Despite the increasing incidence of BM, likely due to consistently improving treatment of primary cancers, BM remain severely understudied. In this study, we utilized patient-derived stem cell lines from lung-to-brain metastases to examine the regulatory role of STAT3 in brain metastasis initiating cells (BMICs). Annotation of our previously described BMIC regulatory genes with protein-protein interaction network mapping identified STAT3 as a novel protein interactor. STAT3 knockdown showed a reduction in BMIC self-renewal and migration, and decreased tumor size in vivo. Screening of BMIC lines with a library of STAT3 inhibitors identified one inhibitor to significantly reduce tumor formation. Meta-analysis identified the oncomir microRNA-21 (miR-21) as a target of STAT3 activity. Inhibition of miR-21 displayed similar reductions in BMIC self-renewal and migration as STAT3 knockdown. Knockdown of STAT3 also reduced expression of known downstream targets of miR-21. Our studies have thus identified STAT3 and miR-21 as cooperative regulators of stemness, migration and tumor initiation in lung-derived BM. Therefore, STAT3 represents a potential therapeutic target in the treatment of lung-to-brain metastases.

Identification of Bidentate Salicylic Acid Inhibitors of PTP1B

PTP1B is a master regulator in the insulin and leptin metabolic pathways. Hyper-activated PTP1B results in insulin resistance and is viewed as a key factor in the onset of type II diabetes and obesity. Moreover, inhibition of PTP1B expression in cancer cells dramatically inhibits cell growth in vitro and in vivo. Herein, we report the computationally guided optimization of a salicylic acid-based PTP1B inhibitor 6, identifying new and more potent bidentate PTP1B inhibitors, such as 20h, which exhibited a > 4-fold improvement in activity. In CHO-IR cells, 20f, 20h, and 20j suppressed PTP1B activity and restored insulin receptor phosphorylation levels. Notably, 20f, which displayed a 5-fold selectivity for PTP1B over the closely related PTPσ protein, showed no inhibition of PTP-LAR, PRL2 A/S, MKPX, or papain. Finally, 20i and 20j displayed nanomolar inhibition of PTPσ, representing interesting lead compounds for further investigation.

Signal transducer and activator of transcription 3 (STAT3) inhibitor, S3I-201, acts as a potent and non-selective alkylating agent

The Signal Transducer and Activator of Transcription 3 (STAT3) oncogene is a master regulator of many human cancers, and a well-recognized target for therapeutic intervention. A well known STAT3 inhibitor, S3I-201 (NSC 74859), is hypothesized to block STAT3 function in cancer cells by binding the STAT3 SH2 domain and disrupt STAT3 protein complexation events. In this study, liquid chromatography tandem mass spectrometry analysis revealed that STAT3, in the presence of S3I-201, showed a minimum of five specific sites of modification, cysteines 108, 259, 367, 542, and 687. Moreover, a prepared fluorescently labeled chemical probe of S3I-201 (DB-6-055) revealed that S3I-201 non-specifically and globally alkylated intracellular proteins at concentrations consistent with S3I-201s reported IC50. These data are consistent with the hypothesis that S3I-201 is a sub-optimal probe for interrogating STAT3-related cell biology.

A selective inhibitor of the UFM1-activating enzyme, UBA5.

Protein conjugation with ubiquitin and ubiquitin-like small molecules, such as UFM1, is important for promoting cancer cell survival and proliferation. Herein, the development of the first selective micromolar inhibitor of the UBA5 E1 enzyme that initiates UFM1 protein conjugation is described. This organometallic inhibitor incorporates adenosine and zinc(II)cyclen within its core scaffold and inhibits UBA5 noncompetitively and selectively over other E1 enzymes and a panel of human kinases. Furthermore, this compound selectively impedes the cellular proliferation (above 50μM) of cancer cells containing higher levels of UBA5. This inhibitor may be used to further probe the intracellular role of the UFM1 pathway in disease progression.

Abstract LB-C19: The investigation of structural determinants that mediate selective inhibition of UBA5, the UFM1 activating enzyme

Protein conjugation with the UFM1 small protein modifier promotes cellular survival in times of ER stress, which is a common occurrence within diseased cells. We have developed the first selective inhibitor of UBA5, the E1 activating enzyme responsible for initiating UFM1 conjugation. Based on the crystal structure of human UBA5, we identified acidic residues in close proximity to the ATP pocket that we chose to target using a zinc-coordinated polyazamacrocycle. This coordination complex was appended to adenosine through alkyl linkers of varying length in order to probe for optimal inhibition of UBA5 activity. Through this small structure-activity relationship (SAR), we identified our lead inhibitor 5C-Z that disrupts UBA5-mediated UFM1 conjugation in vitro through a non-competitive mode of action (IC50 = 4.1 μM, 95% Confidence Interval (C.I.) = 2.1-7.7 μM). Our compound demonstrates selectivity for UBA5 over other E1 enzymes in vitro, as well as over nearly 100 human kinases as evaluated using a kinome screen, despite containing adenosine within its structure. Currently, we are investigating the structural determinants that are indispensible for inhibitory activity. We are making iterative changes by varying each component of 5C-Z including the metal centre, the polyazamacrocycle, the linker, the ribose as well as the nitrogenous base. To date, we have shown that other transition metals are not as active as the zinc (II) centre, increasing the size of the macrocycle abolishes activity and reducing the linkage from the polyazamacrocycle to the linker also significantly abrogates activity. Investigating the role of each functional group on 5C-Z will help delineate the mechanism by which it selectively inhibits UBA5 activity and may shed light in progressing towards a more drug-like small molecule inhibitor. Citation Format: Stacey-Lynn Paiva, Sara da Silva, Mathew Bancerz, Mulu Geletu, Andrew M. Lewis, Jijun Chen, Yafei Cai, Julie L. Lukkarila, Honglin Li, Patrick T. Gunning. The investigation of structural determinants that mediate selective inhibition of UBA5, the UFM1 activating enzyme. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C19.

Abstract LB-A13: Cellular characterization of the selective inhibition of UBA5 by organometallic, adenosine-based inhibitors

Cells that undergo higher protein turnover, such as pancreatic secretory cells and cancerous cells, have the propensity to undergo endoplasmic reticulum (ER) stress. If left uncorrected, ER stress can result in the initiation of apoptosis. To avoid these fates, cells have developed support systems to counteract the apoptotic effects of ER stress, such as conjugation of certain stress-associated proteins with the ubiquitin-fold modifier 1 (UFM1) ubiquitin-like protein. Our research has recently focused on the discovery and in vitro validation of the first selective inhibitor of the UFM1 pathway, 5C-Z, which selectively targets the UFM1 E1 enzyme (UBA5) over other related enzymes and a panel of over 90 human kinases. This novel strategy of inhibiting UBA5 and subsequent UFMylation would make cancer cells that are highly dependent on this system more susceptible to pharmacological disturbances in ER homeostasis, which could lead to the use of milder drug dosing strategies. Our current efforts are focused on studying the effects of 5C-Z on intracellular signalling, the cellular distribution of proteins in the UFM1 pathway, and phenotypic changes within a lung cancer cell lung line (Sk-Luci6). Treatment of lung cancer cells that exhibit high levels of UBA5 protein expression results in decreased cell proliferation (EC50 = 216.9 μM, 95% C.I. = 211.5 - 222.5 μM), yet does not induce cell death in other diseased (A549) or healthy (MRC9) lung cells (up to 200 μM). The decreased cellular proliferation upon 5C-Z treatment mirrors the effect of treatment with UBA5 siRNA. Using immunocytochemistry, it appears that treatment with 5C-Z also induces changes in both cellular morphology and the distribution of UBA5-like staining within the cell. Furthermore, there is an apparent decrease in UFM1-like co-localization with its E2 conjugating enzyme (UFC1) after prolonged incubation with 5C-Z, although further intracellular analysis is required to confirm this observation. Preliminary work also indicates that these cellular effects may also be selective against the UFM1 pathway compared to related ubiquitin-like labelling pathways. We are currently evaluating the effects of 5C-Z on ER stress signalling pathways connected to the UFM1 labelling system. Citation Format: Sara R. da Silva, Mulu Geletu, Fiza Javed, Stacey-Lynn Paiva, Andrew M. Lewis, Honglin Li, Patrick T. Gunning. Cellular characterization of the selective inhibition of UBA5 by organometallic, adenosine-based inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A13.