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1.  A pan-BCL2 inhibitor renders bone marrow resident human leukemia stem cells sensitive to tyrosine kinase inhibition 
Cell stem cell  2013;12(3):316-328.
Leukemia stem cells (LSC) play a pivotal role in chronic myeloid leukemia (CML) tyrosine kinase inhibitor (TKI) resistance and progression to blast crisis (BC), in part, through alternative splicing of self-renewal and survival genes. To elucidate splice isoform regulators of human BC LSC maintenance, we performed whole transcriptome RNA sequencing; splice isoform-specific qRT-PCR, nanoproteomics, stromal co-culture and BC LSC xenotransplantation analyses. Cumulatively, these studies show that alternative splicing of multiple pro-survival BCL2 family genes promotes malignant transformation of myeloid progenitors into BC LSC that are quiescent in the marrow niche and contribute to therapeutic resistance. Notably, a novel pan-BCL2 inhibitor, sabutoclax, renders marrow niche-resident BC LSC sensitive to TKIs at doses that spare normal progenitors. These findings underscore the importance of alternative BCL2 family splice isoform expression in BC LSC maintenance and suggest that combinatorial inhibition of pro-survival BCL2 family proteins and BCR-ABL may eliminate dormant LSC and obviate resistance.
PMCID: PMC3968867  PMID: 23333150
2.  HTS by NMR of Combinatorial Libraries: A Fragment-Based Approach to Ligand Discovery 
Chemistry & biology  2013;20(1):19-33.
Fragment-based ligand design (FBLD) approaches have become more widely used in drug discovery projects from both academia and industry, and are even often preferred to traditional high-throughput screening (HTS) of large collection of compounds (>105). A key advantage of FBLD approaches is that these often rely on robust biophysical methods such as NMR spectroscopy for detection of ligand binding, hence are less prone to artifacts that too often plague the results from HTS campaigns. In this article, we introduce a screening strategy that takes advantage of both the robustness of protein NMR spectroscopy as the detection method, and the basic principles of combinatorial chemistry to enable the screening of large libraries of fragments (>105 compounds) preassembled on a common backbone. We used the method to identify compounds that target protein-protein interactions.
PMCID: PMC3966493  PMID: 23352136
3.  Targeting the Bcl-2 Family for Cancer Therapy 
Programmed cell death is well-orchestrated process regulated by multiple pro-apoptotic and anti-apoptotic genes, particularly those of the Bcl-2 gene family. These genes are well documented in cancer with aberrant expression being strongly associated with resistance to chemotherapy and radiation.
Areas covered
This review focuses on the resistance induced by the Bcl-2 family of anti-apoptotic proteins and current therapeutic interventions currently in preclinical or clinical trials that target this pathway. Major resistance mechanisms that are regulated by Bcl-2 family proteins and potential strategies to circumvent resistance are also examined. Although antisense and gene therapy strategies are used to nullify Bcl-2 family proteins, recent approaches use small molecule inhibitors and peptides. Structural similarity of the Bcl-2 family of proteins greatly favors development of inhibitors that target the BH3 domain, called BH3 mimetics.
Expert opinion
Strategies to specifically identify and inhibit critical determinants that promote therapy-resistance and tumor progression represent viable approaches for developing effective cancer therapies. From a clinical perspective, pretreatment with novel, potent Bcl-2 inhibitors either alone or in combination with conventional therapies hold significant promise for providing beneficial clinical outcomes. Identifying small molecule inhibitors with broader and higher affinities for inhibiting all of the Bcl-2 pro-survival proteins will facilitate development of superior cancer therapies.
PMCID: PMC3955095  PMID: 23173842
BH3 domain; apoptosis; Mcl-1; radiation resistance; chemotherapy resistance
4.  Raf kinase inhibitor RKIP inhibits MDA-9/syntenin-mediated metastasis in melanoma 
Cancer research  2012;72(23):6217-6226.
Melanoma differentiation associated gene-9 (MDA-9), also known as syntenin, functions as a positive regulator of melanoma progression and metastasis. In contrast, the Raf kinase inhibitor RKIP, a negative modulator of RAF-stimulated MEKK activation, is strongly downregulated in metastatic melanoma cells. In this study, we explored an hypothesized inverse relationship between MDA-9 and RKIP in melanoma. Tumor array and cell line analyses confirmed an inverse relationship between expression of MDA-9 and RKIP during melanoma progression. We found that MDA-9 transcriptionally downregulated RKIP in support of a suggested crosstalk between these two proteins. Further, MDA-9 and RKIP physically interacted in a manner that correlated with a suppression of FAK and c-Src phosphorylation, crucial steps necessary for MDA-9 to promote FAK/c-Src complex formation and initiate signaling cascades that drive the MDA-9-mediated metastatic phenotype. Lastly, ectopic RKIP expression in melanoma cells overrode MDA-9-mediated signaling, inhibiting cell invasion, anchorage-independent growth and in vivo dissemination of tumor cells. Taken together, these findings establish RKIP as an inhibitor of MDA-9-dependent melanoma metastasis, with potential implications for targeting this process therapeutically.
PMCID: PMC3939082  PMID: 23066033
RKIP; MDA-9/syntenin, melanoma; c-Src; FAK
5.  MDA-9/Syntenin and IGFBP-2 Promote Angiogenesis in Human Melanoma 
Cancer research  2012;73(2):844-854.
Melanoma differentiation associated gene-9 (mda-9/syntenin) encodes an adapter scaffold protein whose expression correlates with and mediates melanoma progression and metastasis. Tumor angiogenesis represents an integral component of cancer metastasis prompting us to investigate a possible role of mda-9/syntenin in inducing angiogenesis. Genetic (gain-of-function and loss-of-function) and pharmacological approaches were employed to modify mda-9/syntenin expression in normal immortal melanocytes, early radial growth phase melanoma and metastatic melanoma cells. The consequence of modifying mda-9/syntenin expression on angiogenesis was evaluated using both in vitro and in vivo assays, including tube formation assays using human vascular endothelial cells, CAM assays and xenograft tumor animal models. Gain-of-function and loss-of-function experiments confirm that MDA-9/syntenin induces angiogenesis by augmenting expression of several pro-angiogenic factors/genes. Experimental evidence is provided for a model of angiogenesis induction by MDA-9/syntenin in which MDA-9/syntenin interacts with the ECM activating Src and FAK resulting in activation by phosphorylation of Akt, which induces HIF-1α. The HIF-1α activates transcription of Insulin Growth Factor Binding Protein-2 (IGFBP-2), which is secreted thereby promoting angiogenesis and further induces endothelial cells to produce and secrete VEGF-A augmenting tumor angiogenesis. Our studies delineate an unanticipated cell non-autonomous function of MDA-9/syntenin in the context of angiogenesis, which may directly contribute to its metastasis-promoting properties. As a result, targeting MDA-9/syntenin or its downstream-regulated molecules may provide a means of simultaneously impeding metastasis by both directly inhibiting tumor cell transformed properties (autonomous) and indirectly by blocking angiogenesis (non-autonomous).
PMCID: PMC3548987  PMID: 23233738
mda-9/syntenin; melanoma; angiogenesis; IGFBP-2; HuVECs; CAM assay
6.  Heterotypic Sam-Sam association between Odin-Sam1 and Arap3-Sam: binding affinity and structural insights 
Arap3 is a phosphatidylinositol 3 kinase effector protein that plays a role as GTP-ase activator (GAP) for Arf6 and RhoA. Arap3 contains a sterile alpha motif (Sam) domain that presents high sequence homology with the Sam domain of the EphA2-receptor (EphA2-Sam); both Arap3-Sam and EphA2-Sam are able to associate with the Sam domain of the lipid phosphatase Ship2 (Ship2-Sam).
Recently, we have reported on a novel interaction between the first Sam domain of Odin (Odin-Sam1), a protein belonging to the ANKS (ANKyrin repeat and Sam domain containing) family, and EphA2-Sam. In the current work we apply Nuclear Magnetic Resonance (NMR) spectroscopy, Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC) to characterize the association between Arap3-Sam and Odin-Sam1. We show that these two Sam domains interact with low micromolar affinity. Moreover, by means of molecular docking techniques, supported by NMR data, we demonstrate that Odin-Sam1 and Arap3-Sam may bind with a topology that is common to several Sam-Sam complexes.
The unveiled structural details form the basis for the design of potential peptide-antagonists, that could be used as chemical tools to investigate functional aspects related to heterotypic Arap3-Sam associations.
PMCID: PMC3631714  PMID: 23239578
Sam Domains; Protein-Protein Interactions; NMR Spectroscopy; Surface Plasmon Resonance; Isothermal Titration Calorimetry
7.  Targeted Delivery of Paclitaxel to EphA2-Expressing Cancer Cells 
YSA is an EphA2-targeting peptide that effectively delivers anti-cancer agents to prostate cancer tumors (1). Here, we report on how we increased the drug-like properties of this delivery system.
Experimental Design
By introducing non-natural amino acids, we have designed two new EphA2 targeting peptides: YNH, where norleucine and homoserine replace the two methionine residues of YSA, and dYNH, where a D-tyrosine replaces the L-tyrosine at the first position of the YNH peptide. We describe the details of the synthesis of YNH and dYNH paclitaxel conjugates (YNH-PTX and dYNH-PTX) and their characterization in cells and in vivo.
dYNH-PTX showed improved stability in mouse serum and significantly reduced tumor size in a prostate cancer xenograft model and also reduced tumor vasculature in a syngeneic orthotopic allograft mouse model of renal cancer compared to vehicle or paclitaxel treatments.
This study reveals that targeting EphA2 with dYNH drug conjugates could represent an effective way to deliver anti-cancer agents to a variety of tumor types.
Translational Relevance
Overexpression of the EphA2 positively correlates with tumor malignancy and poor prognosis. For this reason, EphA2 is an attractive target for cancer cell specific drug delivery. In this study, we report on the development of dYNH, an EphA2 targeting peptide that when coupled to paclitaxel (PTX) has favorable pharmacological properties and possesses powerful anti-tumor activity in vivo. dYNH-PTX may allow for an expanded therapeutic index of paclitaxel as well as precluding the need for complex formulations and long infusion times.
PMCID: PMC3537892  PMID: 23155185
8.  Identification of small molecules interfering with H1N1 influenza A viral replication 
ChemMedChem  2012;7(12):2227-2235.
Successful Influenza A viral replication requires both viral proteins and host cellular factors. Here we utilized a cellular assay to screen for small molecules capable of interfering with any of such necessary viral or cellular components. We employed an established reporter assay assessing influenza viral replication by monitoring the activity of co-expressed luciferase. We screened a diverse chemical compound library, resulting in the identification of compound 7, inhibiting a novel yet elusive target. Quantitative real-time PCR studies confirmed the dose dependent inhibitory activity of compound 7 in a viral replication assay. Furthermore, we showed that compound 7 was effective in rescuing high dose influenza infection in an in vivo mouse model. As oseltamivir-resistant influenza strains emerge, compound 7 could be further investigated as a possible novel scaffold for the development of anti-influenza agents acting on novel targets.
PMCID: PMC3769975  PMID: 23139022
Influenza virus; Drug discovery; Ugi reaction; tetrazole formation
9.  Selected Approaches for Rational Drug Design and High Throughput Screening to Identify Anti-Cancer Molecules 
Structure-based modeling combined with rational drug design, and high throughput screening approaches offer significant potential for identifying and developing lead compounds with therapeutic potential. The present review focuses on these two approaches using explicit examples based on specific derivatives of Gossypol generated through rational design and applications of a cancer-specific-promoter derived from Progression Elevated Gene-3. The Gossypol derivative Sabutoclax (BI-97C1) displays potent anti-tumor activity against a diverse spectrum of human tumors. The model of the docked structure of Gossypol bound to Bcl-XL provided a virtual structure-activity-relationship where appropriate modifications were predicted on a rational basis. These structure-based studies led to the isolation of Sabutoclax, an optically pure isomer of Apogossypol displaying superior efficacy and reduced toxicity. These studies illustrate the power of combining structure-based modeling with rational design to predict appropriate derivatives of lead compounds to be empirically tested and evaluated for bioactivity. Another approach to cancer drug discovery utilizes a cancer-specific promoter as readouts of the transformed state. The promoter region of Progression Elevated Gene-3 is such a promoter with cancer-specific activity. The specificity of this promoter has been exploited as a means of constructing cancer terminator viruses that selectively kill cancer cells and as a systemic imaging modality that specifically visualizes in vivo cancer growth with no background from normal tissues. Screening of small molecule inhibitors that suppress the Progression Elevated Gene-3-promoter may provide relevant lead compounds for cancer therapy that can be combined with further structure-based approaches leading to the development of novel compounds for cancer therapy.
PMCID: PMC3763986  PMID: 22931411
Progression Elevated Gene-3; Sabutoclax; Apogossypol; BI-97C1; Gossypol; AP-1; PEA3; ETV4; E1AF; c-fos; c-jun; Cancer Terminator Virus
10.  Inhibition of melanoma development in the Nras(Q61K)::Ink4a−/− mouse model by the small molecule BI-69A11 
Pigment cell & melanoma research  2012;26(1):136-142.
Despite advances in developing specific inhibitors to BRAF mutant melanomas, to date there are no effective therapies for tumors bearing NRAS mutations, present in approximately 15–20% of human melanomas. Here, we extend earlier studies, demonstrating that the small molecule BI-69A11 inhibits the growth of melanoma cell lines in vitro and in vivo. Gene expression microarray analysis of BI-69A11-responsive melanoma cells revealed the induction of interferon- and cell death-related genes that were associated with responsiveness to BI-69A11. Strikingly, the administration of BI-69A11 inhibited melanoma development in genetically modified mice bearing an inducible form of activated Nras and a deletion of the Ink4a gene (Nras(Q61K)::Ink4a−/−). Biweekly administration of BI-69A11 starting at 10 weeks or as late as 24 weeks after the induction of mutant Nras expression inhibited melanoma development (100% and 36%, respectively). BI-69A11 treatment did not inhibit the development of histiocytic sarcomas, which comprise about 50% of the tumors in this model. Immunofluorescent staining analyses of CD45 revealed increased levels of immune cell infiltration in BI-69A11–treated tumors. Gene expression profiling of BI-69A11-resistant Nras(Q61K)::Ink4a−/− tumors revealed the upregulation of functional gene networks associated with the cytoskeleton, DNA damage response, and small molecule transport. The ability to attenuate development of NRAS mutant melanomas by BI-69A11 even when administered at a late stage of the tumor development, support its further development and clinical assessment.
PMCID: PMC3632643  PMID: 23035722
11.  Enhanced delivery of mda-7/IL-24 using a serotype chimeric adenovirus (Ad.5/3) in combination with the Apogossypol derivative BI-97C1 (Sabutoclax) improves therapeutic efficacy in low CAR colorectal cancer cells 
Journal of Cellular Physiology  2012;227(5):2145-2153.
Adenovirus (Ad)-based gene therapy represents a potentially viable strategy for treating colorectal cancer. The infectivity of serotype 5 adenovirus (Ad.5), routinely used as a transgene delivery vector, is dependent on Coxsackie-adenovirus receptors (CAR). CAR expression is downregulated in many cancers thus preventing optimum therapeutic efficiency of Ad.5-based therapies. To overcome the low CAR problem, a serotype chimerism approach was used to generate a recombinant Ad (Ad.5/3) that is capable of infecting cancer cells via Ad.3 receptors in a CAR-independent manner. We evaluated the improved transgene delivery and efficacy of Ad.5/3 recombinant virus expressing melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), an effective wide-spectrum cancer-selective therapeutic. In low CAR human colorectal cancer cells RKO, wild-type Ad.5 virus expressing mda-7/IL-24 (Ad.5-mda-7) failed to infect efficiently resulting in lack of expression of MDA-7/IL-24 or induction of apoptosis. However, a recombinant Ad.5/3 virus expressing mda-7/IL-24 (Ad.5/3-mda-7) efficiently infected RKO cells resulting in higher MDA-7/IL-24 expression and inhibition of cell growth both in vitro and in nude mice xenograft models. Addition of the novel Bcl-2 family pharmacological inhibitor Apogossypol derivative BI-97C1 (Sabutoclax) significantly augmented the efficacy of Ad.5/3-mda-7. A combination regimen of suboptimal doses of Ad.5/3-mda-7 and BI-97C1 profoundly enhanced cytotoxicity in RKO cells both in vitro and in vivo. Considering the fact that Ad.5-mda-7 has demonstrated significant objective responses in a Phase I clinical trial for advanced solid tumors, Ad.5/3-mda-7 alone or in combination with BI-97C1 would be predicted to exert significantly improved therapeutic efficacy in colorectal cancer patients.
PMCID: PMC3228880  PMID: 21780116
Viral gene therapy; Mcl-1 inhibition; apoptosis induction; anti-tumor activity
12.  Novel MT1-MMP small molecule inhibitors based on insights into hemopexin domain function in tumor growth§ 
Cancer Research  2012;72(9):2339-2349.
Membrane type-1 matrix metalloproteinase (MT1-MMP) is a promising drug target in malignancy. The structure of MT1-MMP includes the hemopexin domain (PEX) that is distinct from and additional to the catalytic domain. Current MMP inhibitors target the conserved active site in the catalytic domain and, as a result, repress the proteolytic activity of multiple MMPs instead of MT1-MMP alone. In our search for non-catalytic inhibitors of MT1-MMP, we compared the pro-tumorigenic activity of wild-type MT1-MMP with a MT1-MMP mutant lacking PEX (ΔPEX). In contrast to MT1-MMP, ΔPEX did not support tumor growth in vivo, and its expression resulted in small fibrotic tumors that contained increased levels of collagen. Because these findings suggested an important role for PEX in tumor growth, we performed an inhibitor screen to identify small molecules targeting the PEX domain of MT1-MMP. Using the Developmental Therapeutics Program (NCI/NIH) virtual ligand screening compound library as a source and the X-ray crystal structure of PEX as a target, we identified and validated a novel PEX inhibitor. Low dosage, intratumoral injections of PEX inhibitor repressed tumor growth and caused a fibrotic, ΔPEX-like tumor phenotype in vivo. Together, our findings provide a preclinical proof-of-principle rationale for the development of novel and selective MT1-MMP inhibitors that specifically target the PEX domain.
PMCID: PMC3342448  PMID: 22406620
MT1-MMP; hemopexin domain; small molecule; tumor growth; migration; type I collagen
Biochemistry  2012;51(10):2136-2145.
The EphA2 receptor plays key roles in many physiological and pathological events including cancer. The process of receptor endocytosis and the consequent degradation have lately attracted attention as possible means of overcoming the negative outcomes of EphA2 in cancer cells and decreasing tumor malignancy. A recent study indicates that Sam (Sterile Alpha Motif) domains of Odin, a member of the ANKS (Ankyrin repeat and sterile alpha motif domain-containing) family of proteins, are important to regulate EphA2 endocytosis. Odin contains two tandem Sam domains (Odin-Sam1 and Sam2).
Herein we report on the NMR solution structure of Odin-Sam1; through a variety of assays (employing NMR, SPR and ITC techniques), we clearly demonstrate that Odin-Sam1 binds to the Sam domain of EphA2 in the low micromolar range. NMR chemical shift perturbation experiments and molecular modeling studies point out that the two Sam domains interact with a head to tail topology characteristic of several Sam-Sam complexes. This binding mode is similar to that we have previously proposed for the association between the Sam domains of the lipid phosphatase Ship2 and EphA2.
This work further validates structural elements relevant for the heterotypic Sam-Sam interactions of EphA2 and provides novel insights for the design of potential therapeutic compounds that can modulate receptor endocytosis.
PMCID: PMC3319784  PMID: 22332920
14.  A novel targeted system to deliver chemotherapeutic drugs to EphA2-expressing cancer cells 
Journal of Medicinal Chemistry  2012;55(5):2427-2436.
The efficacy of anti-cancer drugs is often limited by their systemic toxicities and adverse side effects. We report that the EphA2 receptor is over-expressed preferentially in several human cancer cell lines compared to normal tissues and that an EphA2 targeting peptide (YSAYPDSVPMMS) can be effective in delivering anti-cancer agents to such tumors. Hence, we report on the synthesis and characterizations of a novel EphA2-targeting agent conjugated with the chemotherapeutic drug paclitaxel. We found that the peptide-drug conjugate is dramatically more effective than paclitaxel alone at inhibiting tumor growth in a prostate cancer xenograft model, delivering significantly higher levels of drug to the tumor site. We believe these studies open the way to the development of a new class of therapeutic compounds that exploit the EphA2 receptor for drug delivery to cancer cells.
PMCID: PMC3299084  PMID: 22329578
15.  Endoplasmic Reticulum Membrane Reorganization Is Regulated by Ionic Homeostasis 
PLoS ONE  2013;8(2):e56603.
Recently we described a new, evolutionarily conserved cellular stress response characterized by a reversible reorganization of endoplasmic reticulum (ER) membranes that is distinct from canonical ER stress and the unfolded protein response (UPR). Apogossypol, a putative broad spectrum BCL-2 family antagonist, was the prototype compound used to induce this ER membrane reorganization. Following microarray analysis of cells treated with apogossypol, we used connectivity mapping to identify a wide range of structurally diverse chemicals from different pharmacological classes and established their ability to induce ER membrane reorganization. Such structural diversity suggests that the mechanisms initiating ER membrane reorganization are also diverse and a major objective of the present study was to identify potentially common features of these mechanisms. In order to explore this, we used hierarchical clustering of transcription profiles for a number of chemicals that induce membrane reorganization and discovered two distinct clusters. One cluster contained chemicals with known effects on Ca2+ homeostasis. Support for this was provided by the findings that ER membrane reorganization was induced by agents that either deplete ER Ca2+ (thapsigargin) or cause an alteration in cellular Ca2+ handling (calmodulin antagonists). Furthermore, overexpression of the ER luminal Ca2+ sensor, STIM1, also evoked ER membrane reorganization. Although perturbation of Ca2+ homeostasis was clearly one mechanism by which some agents induced ER membrane reorganization, influx of extracellular Na+ but not Ca2+ was required for ER membrane reorganization induced by apogossypol and the related BCL-2 family antagonist, TW37, in both human and yeast cells. Not only is this novel, non-canonical ER stress response evolutionary conserved but so also are aspects of the mechanism of formation of ER membrane aggregates. Thus perturbation of ionic homeostasis is important in the regulation of ER membrane reorganization.
PMCID: PMC3574070  PMID: 23457590
16.  Targeting zinc finger domains with small molecules: solution structure and binding studies of the RanBP2-type zinc finger of RBM5 
The RNA Binding Motif protein 5 (RBM5), also known as Luca15 or H37, is a component of prespliceosomal complexes, that regulates the alternative splicing of several mRNAs, such as Fas and caspase-2.
The rbm5 gene is located at the 2p21.3 chromosomal region, which is strongly associated with lung cancer and many other cancers. Both increased and decreased levels of RBM5 can play a role in tumor progression. In particular, down-regulation of rbm5 is involved in lung cancer and other cancers upon Ras activation, and, also, represents a molecular signature associated with metastasis in various solid tumors. On the other hand, up-regulation of rbm5 occurs in breast and ovarian cancer. Moreover, RBM5 was also found to be involved in the early stage of the HIV-1 viral cycle, representing a potential target for the treatment of the HIV-1 infection.
While the molecular basis for RNA recognition and ubiquitin interaction have been structurally characterized, small molecules binding this ZF domain that may contribute to characterize their activity and to develop potential therapeutic agents have not been yet reported. Via an NMR screening of a fragment library we identified several binders and the complex of the most promising one, named compound 1, with the RBM5 ZF1 was structurally characterized in solution. Interestingly, the binding mechanism reveals that compound 1 occupies the RNA binding pocket and is therefore able to compete with the RNA to bind RBM5 RanBP2-type ZF domain, as indicated by NMR studies.
PMCID: PMC3408030  PMID: 22162216
NMR; Fragment-based drug discovery; Zn-finger; RanBP2; RBM5; NMR spectroscopy; protein-ligand interactions
17.  Role of Excitatory Amino Acid Transporter-2 (EAAT2) and Glutamate in Neurodegeneration: Opportunities for Developing Novel Therapeutics 
Journal of Cellular Physiology  2011;226(10):2484-2493.
Glutamate is an essential excitatory neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of the major glutamate transporters expressed predominantly in astroglial cells and is responsible for 90% of total glutamate uptake. Glutamate transporters tightly regulate glutamate concentration in the synaptic cleft. Dysfunction of EAAT2 and accumulation of excessive extracellular glutamate has been implicated in the development of several neurodegenerative diseases including Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. Analysis of the 2.5-kb human EAAT2 promoter showed that NF-κB is an important regulator of EAAT2 expression in astrocytes. Screening of approximately 1,040 FDA-approved compounds and nutritionals led to the discovery that many β-lactam antibiotics are transcriptional activators of EAAT2 resulting in increased EAAT2 protein levels. Treatment of animals with ceftriaxone (CEF), a β-lactam antibiotic, led to an increase of EAAT2 expression and glutamate transport activity in the brain. CEF has neuroprotective effects in both in vitro and in vivo models based on its ability to inhibit neuronal cell death by preventing glutamate excitotoxicity. CEF increases EAAT2 transcription in primary human fetal astrocytes (PHFA) through the NF-κB signaling pathway. The NF-κB binding site at −272 position was critical in CEF-mediated EAAT2 protein induction. These studies emphasize the importance of transcriptional regulation in controlling glutamate levels in the brain. They also emphasize the potential utility of the EAAT2 promoter for developing both low and high throughput screening assays to identify novel small molecule regulators of glutamate transport with potential to ameliorate pathological changes occurring during and causing neurodegeneration.
PMCID: PMC3130100  PMID: 21792905
18.  A Disalicylic Acid-Furanyl Derivative Inhibits Ephrin Binding to a Subset of Eph Receptors 
Chemical biology & drug design  2011;78(4):667-678.
Eph receptor tyrosine kinases and ephrin ligands control many physiological and pathological processes, and molecules interfering with their interaction are useful probes to elucidate their complex biological functions. Moreover, targeting Eph receptors might enable new strategies to inhibit cancer progression and pathological angiogenesis as well as promote nerve regeneration. Because our previous work suggested the importance of the salicylic acid group in antagonistic small molecules targeting Eph receptors, we screened a series of salicylic acid derivatives to identify novel Eph receptor antagonists. This identified a disalicylic acid-furanyl derivative that inhibits ephrin-A5 binding to EphA4 with an IC50 of 3 μM in ELISA assays. This compound, which appears to bind to the ephrin-binding pocket of EphA4, also targets several other Eph receptors. Furthermore, it inhibits EphA2 and EphA4 tyrosine phosphorylation in cells stimulated with ephrin while not affecting phosphorylation of EphB2, which is not a target receptor. In endothelial cells, the disalicylic acid-furanyl derivative inhibits EphA2 phosphorylation in response to TNFα and capillary-like tube formation on Matrigel, two effects that depend on EphA2 interaction with endogenous ephrin-A1. These findings suggest that salicylic acid derivatives could be used as starting points to design new small molecule antagonists of Eph receptors.
PMCID: PMC3196665  PMID: 21791013
small molecule; antagonist; dymethylpyrrole derivative; protein tyrosine kinase; angiogenesis; nerve regeneration
19.  Targeting Mcl-1 for the therapy of cancer 
Human cancers are genetically and epigenetically heterogeneous and have the capacity to commandeer a variety of cellular processes to aid in their survival, growth and resistance to therapy. One strategy is to overexpress proteins that suppress apoptosis, such as the Bcl-2 family protein Mcl-1. The Mcl-1 protein plays a pivotal role in protecting cells from apoptosis and is overexpressed in a variety of human cancers.
Areas covered
Targeting Mcl-1 for extinction in these cancers, using genetic and pharmacological approaches, represents a potentially effectual means of developing new efficacious cancer therapeutics. Here we review the multiple strategies that have been employed in targeting this fundamental protein, as well as the significant potential these targeting agents provide in not only suppressing cancer growth, but also in reversing resistance to conventional cancer treatments.
Expert Opinion
We discuss the potential issues that arise in targeting Mcl-1 and other Bcl-2 anti-apoptotic proteins, as well problems with acquired resistance. The application of combinatorial approaches that involve inhibiting Mcl-1 and manipulation of additional signaling pathways to enhance therapeutic outcomes is also highlighted. The ability to specifically inhibit key genetic/epigenetic elements and biochemical pathways that maintain the tumor state represent a viable approach for developing rationally based, effective cancer therapies.
PMCID: PMC3205956  PMID: 21851287
20.  Design and characterization of a potent and selective dual ATP- and substrate-competitive sub-nanomolar bi-dentate c-Jun N-terminal Kinase (JNK) inhibitor 
Journal of medicinal chemistry  2011;54(18):6206-6214.
c-Jun N-terminal Kinases (JNKs) represent valuable targets in the development of new therapies. Present on the surface of JNK is a binding pocket for substrates and the scaffolding protein JIP1 in close proximity to the ATP binding pocket. We propose that bi-dentate compounds linking the binding energies of weakly interacting ATP and substrate mimetics could result in potent and selective JNK inhibitors. We describe here a bi-dentate molecule, 19, designed against JNK. 19 inhibits JNK kinase activity (IC50 = 18 nM; Ki = 1.5 nM) and JNK/substrate association in a displacement assay with a substrate peptide (compound 20; IC50 = 46 nM; Ki = 2 nM). Our data demonstrate that 19 targets for the ATP and substrate-binding sites on JNK concurrently. Finally, compound 19 not only inhibits JNK in a variety of cell-based experiments, but it elicits also in vivo activity where it is shown to improve glucose tolerance in diabetic mice.
PMCID: PMC3174326  PMID: 21815634
21.  Functional Specialization in Proline Biosynthesis of Melanoma 
PLoS ONE  2012;7(9):e45190.
Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C), and then to proline via pyrroline-5-carboxylate reductases (PYCRs). Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of 13C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis.
PMCID: PMC3443215  PMID: 23024808
22.  SAR by Inter-Ligand nuclear Overhauser Effects (ILOEs) based discovery of acylsulfonamide compounds active against Bcl-xL and Mcl-1 
Journal of medicinal chemistry  2011;54(17):6000-6013.
Over expression of anti-apoptotic members of the Bcl-2 family proteins, such as Bcl-xL and Mfl-1 has been shown to be involved in resistance to chemotherapeutic drugs in many forms of cancers. Recent efforts from the Abbott Laboratories resulted in the development of the acylsulfonamide compound and clinical candidate that targets selectively Bcl-2, Bcl-xL and Bcl-w while is not active against Mcl-1 and Bfl-1. However, early clinical and pre-clinical studies suggest that pan-Bcl-2 antagonists, targeting simultaneously Mcl-1, Bcl-xL and possibly all other four anti-apoptotic Bcl-2 proteins, may result in more efficacious drugs. Here, following an NMR fragment-based approach, SAR by ILOEs, we report on compounds that exhibit nanomolar affinities for both BclxL and Mcl-1 in vitro. We believe that these molecules can be used as useful starting point for the development of novel Bcl-2 antagonists, in particular targeting Mcl-1.
PMCID: PMC3165075  PMID: 21797225
23.  Targeting Metallo-proteins by Fragment-Based Lead Discovery 
Chemical biology & drug design  2011;78(2):211-223.
It has been estimated that nearly one third of functional proteins contain a metal ion. These constitute a wide variety of possible drug targets including metalloproteinases, dehydrogenases, oxidoreductases, hydrolases, deacetylases or many others in which the metal ion is either of catalytic or structural nature. Despite the predominant role of a metal ion in so many classes of drug targets, current high-throughput screening techniques do not usually produce viable hits against these proteins, likely due to the lack of proper metal binding pharmacophores in the current screening libraries. Herein we describe a novel fragment based drug discovery approach using a metal targeting fragment library that is based on a variety of distinct classes of metal-binding groups designed to reliably anchor the fragments at the target's metal ions. We show that the approach can effectively identify novel, potent and selective agents that can be readily developed into metalloprotein-targeted therapeutics.
PMCID: PMC3135788  PMID: 21564556
24.  Effective inhibition of melanoma by BI-69A11 is mediated by dual targeting of the AKT and NF-κB pathways 
Pigment cell & melanoma research  2011;24(4):703-713.
In melanoma, the activation of pro-survival signaling pathways, such as the AKT and NF-κB pathways, are critical for tumor growth. We have recently reported that the AKT inhibitor BI-69A11 causes efficient inhibition of melanoma growth. Here, we show that in addition to its AKT inhibitory activity, BI-69A11 also targets the NF-κB pathway. In melanoma cell lines, BI-69A11 inhibited TNF-α-stimulated IKKα/β and IκB phosphorylation as well as NF-κB reporter gene expression. Furthermore, the effective inhibition of melanoma growth by BI-69A11 was attenuated upon NF-κB activation. Mechanistically, reduced NF-κB signaling by BI-69-A11 is mediated by the inhibition of sphingosine kinase 1, identified in a screen of 315 kinases. Significantly, we demonstrate that BI-69A11 is well-tolerated and orally active against UACC 903 and SW1 melanoma xenografts. Our results demonstrate that BI-69A11 inhibits both the AKT and NF-κB pathways and that the dual targeting of these pathways may be efficacious as a therapeutic strategy in melanoma.
Although B-RAF and MEK inhibitors have shown promise in the clinic against melanoma, the development of resistance to these singly targeted agents inevitably results. These observations underscore the plasticity of melanoma to chemotherapeutic agents and further emphasize the need to apply combinatorial targeting of signaling pathways as a strategy to maximize therapeutic response. The PI3K/AKT and NF-κB signaling pathways are altered in melanoma, presenting additional opportunities for target inhibition. Our studies demonstrate that the AKT inhibitor, BI-69A11, also inhibits the NF-κB pathway and that dual inhibition of both pathways is responsible for the anti-tumor efficacy of this molecule.
PMCID: PMC3158838  PMID: 21592316
melanoma; AKT; NF-kB; targeted therapy
25.  Sabutoclax, a Mcl-1 Antagonist, Inhibits Tumorigenesis in Transgenic Mouse and Human Xenograft Models of Prostate Cancer12 
Neoplasia (New York, N.Y.)  2012;14(7):656-665.
Resistance to available therapeutic agents has been a common problem thwarting progress in treatment of castrate-resistant and metastatic prostate cancer (PCa). Overexpression of the Bcl-2 family members, including Mcl-1, in PCa cells is known to inhibit intracellular mitochondrial-dependent apoptosis. Here we report the development of a novel transgenic mouse model that spontaneously develops prostatic intraepithelial neoplasia and adenocarcinoma by the inducible, conditional knockout of transforming growth factor β receptor type II in stromal fibroblastic cells (Tgfbr2ColTKO). The Tgfbr2ColTKO prostate epithelia demonstrated down-regulation of luminal and basal differentiation markers, as well as Pten expression and up-regulation of Mcl-1. However, unlike in men, Tgfbr2ColTKO prostates exhibited no regression acutely after castration. The administration of Sabutoclax (BI-97C1), a pan-active Bcl-2 protein family antagonist mediated apoptosis in castrate-resistant PCa cells of Tgfbr2ColTKO mice and human subcutaneous, orthotopic, and intratibial xenograft PCa models. Interestingly, Sabutoclax had little apoptotic effect on benign prostate tissue in Tgfbr2ColTKO and wild-type mice. Sabutoclax was able to block c-Met activation, a critical axis in PCa metastatic progression. Further, Sabutoclax synergistically sensitized PC-3 cells to the cytotoxic effects of docetaxel (Taxotere). Together, these data suggest that Sabutoclax inhibits castrate-resistant PCa alone at the primary and bone metastatic site as well as support sensitivity to docetaxel treatment.
PMCID: PMC3421961  PMID: 22904682

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