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.
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.
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.
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.
NMR; Fragment-based drug discovery; Zn-finger; RanBP2; RBM5; NMR spectroscopy; protein-ligand interactions
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.
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.
small molecule; antagonist; dymethylpyrrole derivative; protein tyrosine kinase; angiogenesis; nerve regeneration
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.
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.
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.
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.
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.
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.
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.
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.
melanoma; AKT; NF-kB; targeted therapy
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.
A new series of 3-ethynyl-1H–indazoles has been synthesized and evaluated in both biochemical and cell-based assays as potential kinase inhibitors. Interestingly, a selected group of compounds identified from this series exhibited low micromolar inhibition against critical components of the PI3K pathway, targeting PI3K, PDK1 and mTOR kinases. Combination of computational modeling and structure-activity relationships studies reveal a possible novel mode for PI3K inhibition, resulting in a PI3Kα isoform specific compound. Hence, by targeting the most oncogenic mutant isoform of PI3K, the compound displays anti-proliferative activity both in monolayer human cancer cell cultures and in three-dimensional tumor models. Because of its favorable physicochemical, in vitro ADME and drug-like properties, we propose that this novel ATP mimetic scaffold could result useful in deriving novel selecting and multi-kinase inhibitors for clinical use.
We report comprehensive structure activity relationship studies on a novel series of c-Jun N-terminal kinase (JNK) inhibitors. Intriguingly, the compounds have a dual inhibitory activity by functioning as both ATP and JIP mimetics, possibly by binding to both the ATP binding site and to the docking site of the kinase. Several of such novel compounds display potent JNK inhibitory profiles both in vitro and in cell.
We report on the design and evaluation of novel cyclic peptides targeting the N-terminal domain of the protein tyrosine phosphatase YopH from Yersinia. Cyclic peptides have been designed based on a short sequence from the protein SKAP-HOM [DE(pY)DDPF (pY = phosphotyrosine)], and they all contain the motif DEZXDPfK (where Z is a phosphotyrosine or a non-hydrolyzable phosphotyrosine mimetic, X is an aspartic acid or a leucine and f is a d-phenylalanine). These peptides present a ‘head to tail’ architecture, enabling cyclization through formation of an amide bond in between the side chains of the first aspartic acid and the lysine residues. Chemical shift perturbation studies have been carried out to monitor the binding of these peptides to the N-terminal domain of YopH. Peptides containing a phosphotyrosine moiety exhibit binding affinities in the low micromolar range; substitution of the phosphotyrosine with one of its non-hydrolyzable derivatives dramatically reduces the binding affinities. These preliminary studies may pave the way for the discovery of more potent and selective peptidebased ligands of the YopH N-terminal domain which could be further investigated for their ability to inhibit Yersiniae infections.
cyclic peptides; NMR; protein tyrosine phosphatase; Yersinia; Yersinia outer protein H
Overexpression of anti-apoptotic Bcl-2 family proteins is commonly related with tumor maintenance, progression, and chemoresistance. Inhibition of these anti-apoptotic proteins is an attractive approach for cancer therapy. Guided by nuclear magnetic resonance (NMR) binding assays, a series of 5, 5′ substituted compound 6a (Apogossypolone) derivatives was synthesized and identified pan-active antagonists of anti-apoptotic Bcl-2 family proteins, with binding potency in the low micromolar to nanomolar range. Compound 6f inhibits the binding of BH3 peptides to Bcl-XL, Bcl-2 and Mcl-1 with IC50 values of 3.10, 3.12 and 2.05 μM, respectively. In a cellular assay, 6f potently inhibits cell growth in several human cancer cell lines in a dose-dependent manner. Compound 6f further displays in vivo efficacy in transgenic mice and demonstrated superior single-agent antitumor efficacy in a PPC-1 mouse xenograft model. Together with its negligible toxicity, compound 6f represents a promising drug lead for the development of novel apoptosis-based therapies for cancer.
Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), a cytokine belonging to the IL-10 family, selectively induces apoptosis in cancer cells without harming normal cells by promoting an endoplasmic reticulum (ER) stress response. The precise molecular mechanism by which the ER stress response culminates in cell death requires further clarification. The present study shows that in prostate carcinoma cells, the mda-7/IL-24-induced ER stress response causes apoptosis by translational inhibition of the antiapoptotic protein myeloid cell leukemia-1 (Mcl-1). Forced expression of Mcl-1 blocked mda-7/IL-24 lethality, whereas RNA interference or gene knockout of Mcl-1 markedly sensitized transformed cells to mda-7/IL-24. Mcl-1 downregulation by mda-7/IL-24 relieved its association with the proapoptotic protein Bak, causing oligomerization of Bak and leading to cell death. These observations show the profound role of the Bcl-2 protein family member Mcl-1 in regulating cancer-specific apoptosis induced by this cytokine. Thus, our studies provide further insights into the molecular mechanism of ER stress-induced cancer-selective apoptosis by mda-7/IL-24. As Mcl-1 is overexpressed in the majority of prostate cancers, mda-7/IL-24 might provide an effective therapeutic for this disease.
This study aims at the identification of novel structural features on the surface of the Zn-dependant metalloprotease Lethal Factor (LF) from Anthrax onto which to design novel and selective inhibitors. We report that by targeting an unexplored region of LF that exhibits ligand-induced conformational changes we could obtain inhibitors with at least 30-fold LF selectivity compared to two other most related human metalloproteases MMP-2 and MMP-9. Based on these results, we propose a novel pharmacophore model that, together with the preliminarily identified compounds, should help the design of more potent and selective inhibitors against Anthrax.
We have recently identified a series of compounds which efficiently inhibit Anthrax lethal factor (LF) metallo-protease. Here we present further structure activity relationship and CoMFA (Comparative Molecular Field Analysis) studies on newly derived inhibitors. The obtained 3D QSAR model was subsequently compared with the X-ray structure of the complex between LF and a representative compound. Our studies form the basis for the rational design of additional compounds with improved activity and selectivity.
We describe the use of a furanyl salicyl nitroxide derivative (“spin-labeled” compound), as a paramagnetic phosphotyrosine mimetic, to carry out a second-site screening by NMR against the PTPase YopH from Yersinia pestis. Using such a fragment-based screening approach we identified several small molecules targeting YopH that bind at sites adjacent to the spin-labeled compound. These second-site fragments were subsequently used to design and synthesize bidentate YopH inhibitors with sub-micromolar in vitro inhibition, selectivity against the human PTPase PTP1B, and cellular activity against Y. Pseudotuberculosis.
These initial compounds could result useful in elucidating the structural determinants necessary for YopH inhibition and may help in the design of even more active, selective and cell permeable compounds for the development of novel therapies against Yersiniae.
Yersinia; NMR screening; spin label; YopH; Protein tyrosine phosphatase
In our continued attempts to identify novel and effective pan-Bcl-2 antagonists, we have recently reported a series of compound 2 (Apogossypol) derivatives, resulting in the chiral compound 4 (8r). We report here on synthesis and evaluation on its optically pure individual isomers. Compound 11 (BI-97C1), the most potent diastereoisomer of compound 4, inhibits the binding of BH3 peptides to Bcl-XL, Bcl-2, Mcl-1 and Bfl-1 with IC50 values of 0.31, 0.32, 0.20 and 0.62 μM, respectively. The compound also potently inhibits cell growth of human prostate cancer, lung cancer and lymphoma cell lines with EC50 values of 0.13, 0.56 and 0.049 μM, respectively and shows little cytotoxicity against bax−/−bak−/− cells. Compound 11 displays in vivo efficacy in transgenic mice models and also demonstrated superior single-agent antitumor efficacy in a prostate cancer mouse xenograft model. Therefore, compound 11 represents a potential drug lead for the development of novel apoptosis-based therapies against cancer.
Persistently hyper-phosphorylated Akt contributes to human oncogenesis and resistance to therapy. TCN-P, the active metabolite of the Akt phosphorylation inhibitor triciribine (TCN), is in clinical trials, but the mechanism by which TCN-P inhibits Akt phosphorylation is unknown. Here we show that in vitro, TCN-P inhibits neither Akt activity nor the phosphorylation of Akt S473 and T308 by mTOR or PDK1, respectively. However, in intact cells, TCN inhibits EGF-stimulated Akt recruitment to the plasma membrane and phosphorylation of Akt. Surface plasmon resonance (SPR) demonstrates that TCN-P, but not TCN, binds Akt-derived pleckstrin homology (PH) domain (KD: 690 nM). Furthermore, nuclear magnetic resonance (NMR) spectroscopy shows that TCN-P, but not TCN, binds to the PH domain in the vicinity of the PIP3 binding pocket. Finally, constitutively active Akt mutants, Akt1-T308D/S473D and myr-Akt1, but not the transforming mutant Akt1-E17K, are resistant to TCN-P and rescue from TCN inhibition of proliferation and induction of apoptosis. Thus, our studies indicate that TCN-P binds to the PH domain of Akt and blocks its recruitment to the membrane and that the subsequent inhibition of Akt phosphorylation contributes to TCN-P anti-proliferative and pro-apoptotic activity, suggesting that this drug may be beneficial to patients whose tumors express persistently phosphorylated Akt.
A chelator fragment library based on a variety of metal binding groups was screened against a metalloproteinase. Lead hits were identified and an expanded library of select compounds was synthesized, resulting in numerous high-affinity hits against several metalloprotein targets. The findings clearly demonstrate that chelators can be used to generate libraries suitable for fragment-based lead design (FBLD) directed at important metalloproteins.
chelators; fragment-based lead design; libraries; metalloproteins; zinc
A series of thiadiazole derivatives has been designed as potential allosteric, substrate competitive inhibitors of the protein kinase JNK. We report on the synthesis, characterization and evaluation of a series of compounds that resulted in the identification of potent and selective JNK inhibitors targeting its JIP-1 docking site.