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We herein report on the pharmacophore determination of the ERK docking domain inhibitor (Z)-3-(2-aminoethyl)-5-(4-ethoxybenzylidene)thiazolidine-2,4-dione, which has led to the discovery of compounds with greater selectivities for inhibiting the proliferation of melanoma cells containing active ERK signaling.

We used fluorescence spectroscopy and surface tensiometry to study the interaction between low-generation (G1 and G4) poly(amidoamine) (PAMAM) dendrimers, potential vehicles for intracellular drug delivery, and model lipid bilayers. Membrane association of fluorescently labeled dendrimers, measured by fluorescence anisotropy, increased with increasing size of the dendrimer and with increasing negative charge density in the membrane, indicating the electrostatic nature of the interaction. When the membrane was doped with pyrene-labeled phosphatidyl glycerol (pyrene-PG), pyrene excimer fluorescence demonstrated a dendrimer-induced selective aggregation of negatively charged lipids when the membrane was in the liquid crystalline state. A nonlinear Stern–Volmer quenching of dendrimer fluorescence with cobalt bromide suggested a dendrimer-induced aggregation of lipid vesicles, which increased with the dendrimer's generation number. Surface tensiometry measurements showed that dendrimers penetrated into the lipid monolayer only at subphysiologic surface pressures (

The extracellular-signal-regulated kinase (ERK) proteins belong to the mitogen-activated protein kinase family. They participate in several important cell-signaling pathways. Unregulated, ERK2 is thought to mediate cell proliferation in many types of cancer. But because ERK also functions in many normal cell activities, inhibition selectivity is necessary for its use in cancer therapy: one needs to block only phosphorylation of those substrates that are involved in cell proliferation, while leaving phosphorylation of other substrates unaffected. Novel small-molecule compounds, which inhibit phosphorylation of selected substrates but do not compete with ATP for its binding site, have been previously identified by computer-aided drug design (CADD) and molecular-dynamics docking algorithms. Herein, we spectroscopically characterized selected compounds, and show that they can serve as resonance energy transfer (RET) acceptors for tryptophan: we determined the values of spectral overlap J between the compounds' absorption and ERK fluorescence, and of the Forster distance R0 for the compound/tryptophan pairs. We then used RET to determine average distances between the bound compounds and the group of three tryptophans in ERK2, thus validating the CADD predictions of the compounds' binding sites. The problem of multiple but closely located donors (tryptophans) that pass the energy to the single acceptor (the bound compound) is discussed, as is the possibility of utilizing RET for mapping the inhibitor-binding sites on other kinases. The novel RET acceptors for tryptophan can conceivably be employed in studies of other protein/ligand systems with suitable spectroscopic properties.

Our study was designed to determine if compounds used experimentally to generate nitric oxide excess differ in ability to elicit degenerative stress to cholinergic neurons and, if so, what mechanisms account for their differences. Nitric oxide donors are often used experimentally in attempts to emulate the bioactivities of endogenous NO, but the pharmacological actions of NO donors can vary dramatically according to the species of NO (NOx) and other agents (e.g., iron cations, cyanide anion, superoxide anion) released, and as affected by the state of the cellular redox environment. To determine whether different types of NO donors exert differential toxicity in a cholinergic neuronal model, we measured cell viability markers, indicators of NOx formation, levels of intracellular-reduced glutathione (GSH), protein nitrosothiols, and the activation of the transcription factor NF-kappaB in a mouse medial septal cholinergic cell line (clone SN56) following exposure to the NO donors S-nitroso-N-acetyl-dl-penicillamine (SNAP), 3-morpholinosydnonimine (SIN-1), or sodium nitroprusside (SNP). SNAP and SIN-1, but not SNP, elicited dramatic increases in media nitrite and intracellular NOx-related fluorescence from cells preloaded with a NOx indicator. Nevertheless, SN56 cells were readily killed by SNP (IC(50) approximately 0.5 mM), while even higher levels (up to 2 mM) of SNAP or SIN-1 were essentially ineffective. SNAP (an NO(+) generator) and SIN-1 (a peroxynitrite generator) both caused increases in SN56 GSH levels; in contrast, SNP caused an immediate and rapid decline in GSH. The increase in GSH in response to SNAP and SIN-1 probably indicates augmentation of intracellular defense mechanisms, because prior depletion of GSH rendered the cells vulnerable to these two donors. GSH depletion did not change the potency of SNP, but GSH depletion made SNAP about twice as potent as SNP. SNAP and SNP, but not SIN-1, activated the transcription factor NF-kappaB, as indicated by increases in p65 nuclear immunoreactivity. Treatment with SNAP, but not SNP or SIN-1, increased levels of S-nitrosothiols in SN56 proteins, consistent with the transfer of an NO(+) equivalent to intracellular thiols. Our experiments show that these three NO donors differ dramatically in their ability to intoxicate SN56 cells, probably because of the different species of NOx and other agents they release, and as reflected in their differing modes of interaction with cellular antioxidant and survival systems.

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are ubiquitously expressed in almost all tissues and cell types. Accordingly, ERK1/2 regulate a diversity of cellular processes by phosphorylating more than 150 substrate proteins. With its constitutive activation being frequently implicated in human malignancies, ERK has been an appealing target in anti-cancer drug development. However, currently available ERK1/2 inhibitors are ATP-competitive. Since these ERK inhibitors block all ERK functions, the potential for side effects in normal tissues and cells may limit their potential of being developed into therapeutic agents. To overcome this limitation, we used a combination of computational and experimental methods to develop small molecular weight inhibitors that target ERK-substrate docking domains. Here we report the identification, characterization and optimization of a compound with a thienyl benzenesulfonate scaffold that specifically inhibits substrates containing an F-site or DEF (docking site for ERK, FXF) motif. Our X-ray crystallography data showed that this compound bound to a pocket in the vicinity of the F-site docking domain on ERK. Biological analysis further demonstrated that this compound and its analogs could preferentially inhibit phosphorylation and function of F-site-containing ERK substrates, and that these compounds preferentially inhibited growth of melanoma cells harboring a V600E B-Raf mutant. Additionally, this compound was shown to inhibit the phosphorylation of specific PKC isoforms via an ERK dependent mechanism. These findings represent the first identification and validation of chemicals that selectively inhibit ERK substrates and signaling events through ATP-independent mechanisms. These compounds have potential utility for elucidating the complex biological roles of ERK1/2 and development into novel anti-cancer agents. Citation Format: Jun Zhang, Taiji Oashi, Ramin Samadani, Kerrick Nevels, Kimberly Burkhard, Deva Priyakumar, Prabhu Raman, Steven Fletcher, Edvin Pozharsky, Paul Shapiro, Alexander MacKerell. A docking domain directed inhibitor targeting ERK regulated F-site substrates. [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 4554. doi:10.1158/1538-7445.AM2013-4554

Extracellular signal-related kinase-1 and 2 (ERK1/2) are hyperactivated in several human cancers, predominantly due to gain-of function mutations in the upstream Ras and Raf proteins. However, its complete inhibition through targeting its active site is expected to be detrimental, since ERK is crucial in various “housekeeping” roles in normal cells. Therefore, our aim is to block specific substrate docking domains on the surface of ERK and, hence, inhibit functions involved in cancer cell proliferation, such as the phosphorylation (activation) of the transcription factor Elk-1 and the enzyme ribosomal S6 kinase-1 (RSK-1). We have previously used computer-aided drug design (CADD) to identify ERK docking domain inhibitors, which led to the discovery of (Z)-3-(2-aminoethyl)-5-(4-ethoxybenzylidene)thiazolidine-2,4-dione (referred to in the literature as 76) that is believed to target the groove in between the common docking (CD) and ED domains. A fluorescence quenching assay showed that 76 binds ERK2 with a KD of 5 μM, exhibits minimal selectivity over the structurally related p38α MAP kinase, and inhibits the proliferation of A375 melanoma cells, which harbor constitutive ERK pathway activation owing to a B-Raf mutation, with an IC50 value of about 20 μM. Through functionalization of the primary amino group of our lead to afford a focused library of amides, carbamates, sulfonamides and anilines, we have been able to improve binding affinities for ERK2 to as low as 70 nM. Importantly, selectivity for ERK2 over p38α was increased to more than 70-fold. Mutation studies suggested that several of our best compounds bind in the vicinity of Thr157 (located at the ED domain) and, to a lesser extent, Asp319 of the CD domain (a residue that was predicted by CADD to bind the primary amino group of 76). Immunoblot analysis demonstrated that new lead SF-2-054 (ERK2: KD = 170 nM) is a more potent inhibitor of Elk-1 phosphorylation than the parent compound 76. Furthermore, negligible effects on the levels of activated ERK2 suggested that SF-2-054 does not operate upstream of ERK2. Consistent with the mutation binding data, SF-2-054 exhibits minimal effects of the levels of phosphorylated Rsk-1, which is known to bind the CD site, opening the door for the development of substrate-specific ERK inhibitors. At the same time, it is noteworthy that this data suggests that SF-2-054 does not bind the catalytic domain of ERK2. In addition, SF-2-054 was found to inhibit proliferation of A375 cells with an IC50 value of < 5 μM, around 5 times more potent than in the HeLa cell line. In contrast to B-Raf (and MEK) inhibitors that afford complete blockade of the B-Raf/MEK/ERK signaling pathway, our approach demonstrates that only partial inhibition of ERK signaling function is necessary to inhibit the proliferation of melanoma cells. Moreover, it is envisaged that targeting ERK docking sites will lead to inhibitors with improved selectivities to which the development of resistance is less likely. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B132.