Structural insights reveal a recognition feature for tailoring hydrocarbon stapled-peptides against the eukaryotic translation initiation factor 4E protein.

Stapled-peptides have emerged as an exciting class of molecules which can modulate protein-protein interactions. We have used a structure-guided approach to rationally develop a set of hydrocarbon stapled-peptides with high binding affinities and residence times against the oncogenic eukaryotic translation initiation factor 4E (eIF4E) protein. Crystal structures of these peptides in complex with eIF4E show that they form specific interactions with a region on the protein-binding interface of eIF4E which is distinct from the other well-established canonical interactions. This recognition element is a major molecular determinant underlying the improved binding kinetics of these peptides with eIF4E. The interactions were further exploited by designing features in the peptides to attenuate disorder and increase helicity which collectively resulted in the generation of a distinct class of hydrocarbon stapled-peptides targeting eIF4E. This study details new insights into the molecular basis of stapled-peptide: eIF4E interactions and their exploitation to enhance promising lead molecules for the development of stapled-peptide compounds for oncology.

In1-ghrelin splicing variant is associated with reduced disease-free survival of breast cancer patients and increases malignancy of breast cancer cells lines.

Ghrelin gene generates several variants that regulate multiple pathophysiological functions, including tumor-related processes. In1-ghrelin is a splicing variant that was previously shown to be overexpressed in breast cancer (BCa), where it correlated with proliferation markers; however, its possible association with clinical outcome of BCa patients and underlying mechanisms are still unknown. To address this issue, expression levels and clinical associations of In1-ghrelin were analyzed in a cohort of 117 BCa samples. Additionally, a battery of cellular and molecular assays was implemented using two BCa cell lines (MCF-7 and MDA-MB-231), wherein the role of In1-ghrelin on proliferation, migration, dedifferentiation and signaling pathways was explored. The results generated revealed that high expression of In1-ghrelin in BCa samples was associated with lymph node metastasis and reduced disease-free survival. Indeed, In1-ghrelin overexpression stimulated proliferation and migration in MCF-7 and MDA-MB-231 cells. Similar results were found by treating MDA-MB-231 and MCF-7 with In1-ghrelin-derived peptides. Conversely, In1-ghrelin silencing decreased proliferation and migration capacities of MDA-MB-231. Furthermore, In1-ghrelin (but not ghrelin) overexpression increased the capacity to form mammospheres in both cell lines. These effects could be associated with activation of MAPK-ERK, Jag1/Notch, Wnt/ß-catenin and/or TGF-ß1 pathways. Altogether, our data indicate that In1-ghrelin could play relevant functional roles in the regulation of BCa development and progression and may provide insights to identify novel biomarkers and new therapeutic approaches for this pathology.

The oncogenic role of the In1-ghrelin splicing variant in prostate cancer aggressiveness.

BACKGROUND: The Ghrelin-system is a complex, pleiotropic family composed of several peptides, including native-ghrelin and its In1-ghrelin splicing variant, and receptors (GHSR 1a/b), which are dysregulated in various endocrine-related tumors, where they associate to pathophysiological features, but the presence, functional role, and mechanisms of actions of In1-ghrelin splicing variant in prostate-cancer (PCa), is completely unexplored. Herein, we aimed to determine the presence of key ghrelin-system components (native-ghrelin, In1-ghrelin, GHSR1a/1b) and their potential pathophysiological role in prostate cancer (PCa). METHODS: In1-ghrelin and native-ghrelin expression was evaluated by qPCR in prostate tissues from patients with high PCa-risk (n = 52; fresh-tumoral biopsies), and healthy-prostates (n = 12; from cystoprostatectomies) and correlated with clinical parameters using Spearman-test. In addition, In1-ghrelin and native-ghrelin was measured in plasma from an additional cohort of PCa-patients with different risk levels (n = 30) and control-healthy patients (n = 20). In vivo functional (proliferation/migration) and mechanistic (gene expression/signaling-pathways) assays were performed in PCa-cell lines in response to In1-ghrelin and native-ghrelin treatment, overexpression and/or silencing. Finally, tumor progression was monitored in nude-mice injected with PCa-cells overexpressing In1-ghrelin, native-ghrelin and empty vector (control). RESULTS: In1-ghrelin, but not native-ghrelin, was overexpressed in high-risk PCa-samples compared to normal-prostate (NP), and this expression correlated with that of PSA. Conversely, GHSR1a/1b expression was virtually absent. Remarkably, plasmatic In1-ghrelin, but not native-ghrelin, levels were also higher in PCa-patients compared to healthy-controls. Furthermore, In1-ghrelin treatment/overexpression, and to a much lesser extent native-ghrelin, increased aggressiveness features (cell-proliferation, migration and PSA secretion) of NP and PCa cells. Consistently, nude-mice injected with PC-3-cells stably-transfected with In1-ghrelin, but not native-ghrelin, presented larger tumors. These effects were likely mediated by ERK1/2-signaling activation and involved altered expression of key oncogenes/tumor suppressor genes. Finally, In1-ghrelin silencing reduced cell-proliferation and PSA secretion from PCa cells. CONCLUSIONS: Altogether, our results indicate that In1-ghrelin levels (in tissue) and circulating levels (in plasma) are increased in PCa where it can regulate key pathophysiological processes, thus suggesting that In1-ghrelin may represent a novel biomarker and a new therapeutic target in PCa.

In1-ghrelin splicing variant is overexpressed in pituitary adenomas and increases their aggressive features.

Pituitary adenomas comprise a heterogeneous subset of pathologies causing serious comorbidities, which would benefit from identification of novel, common molecular/cellular biomarkers and therapeutic targets. The ghrelin system has been linked to development of certain endocrine-related cancers. Systematic analysis of the presence and functional implications of some components of the ghrelin system, including native ghrelin, receptors and the recently discovered splicing variant In1-ghrelin, in human normal pituitaries (n = 11) and pituitary adenomas (n = 169) revealed that expression pattern of ghrelin system suffers a clear alteration in pituitary adenomasas compared with normal pituitary, where In1-ghrelin is markedly overexpressed. Interestingly, in cultured pituitary adenoma cells In1-ghrelin treatment (acylated peptides at 100 nM; 24-72 h) increased GH and ACTH secretion, Ca(2+) and ERK1/2 signaling and cell viability, whereas In1-ghrelin silencing (using a specific siRNA; 100 nM) reduced cell viability. These results indicate that an alteration of the ghrelin system, specially its In1-ghrelin variant, could contribute to pathogenesis of different pituitary adenomas types, and suggest that this variant and its related ghrelin system could provide new tools to identify novel, more general diagnostic, prognostic and potential therapeutic targets in pituitary tumors.

Peptide therapeutics: targeting the undruggable space.

Rapid advancements in genomics have brought a better understanding of molecular mechanisms for various pathologies and identified a number of highly attractive target classes. Some of these targets include intracellular protein-protein interactions (PPIs), which control many essential biological pathways. Their surfaces are part of a diverse and unexplored biological space, where traditional small molecule scaffolds are not always successful. While large biologics can effectively modulate PPIs in the extracellular region, their limitation in crossing the cellular membrane leaves intracellular protein targets outside of their reach. There is a growing need in the pharmaceutical field to push the boundaries of traditional drug design and discover innovative molecules that are able to modulate key biological pathways by inhibiting intracellular PPIs. Peptides are one of the most promising classes of molecules that could deliver such therapeutics in the near future. In this review, we describe technological advancements and emerging chemical approaches for stabilizing active peptide conformations, including stapling, hydrogen bond surrogates, beta-hairpin mimetics, grafting on stable scaffolds, and macrocyclization. These design strategies carry the promise of opening the doors for peptide therapeutics to reach the currently "undruggable" space.

Structural analysis of the human cannabinoid receptor one carboxyl-terminus identifies two amphipathic helices.

Recent research has implicated the C-terminus of G-protein coupled receptors in key events such as receptor activation and subsequent intracellular sorting, yet obtaining structural information of the entire C-tail has proven a formidable task. Here, a peptide corresponding to the full-length C-tail of the human CB1 receptor (residues 400-472) was expressed in E.coli and purified in a soluble form. Circular dichroism (CD) spectroscopy revealed that the peptide adopts an alpha-helical conformation in negatively charged and zwitterionic detergents (48-51% and 36-38%, respectively), whereas it exhibited the CD signature of unordered structure at low concentration in aqueous solution. Interestingly, 27% helicity was displayed at high peptide concentration suggesting that self-association induces helix formation in the absence of a membrane mimetic. NMR spectroscopy of the doubly labeled ((15)N- and (13)C-) C-terminus in dodecylphosphocholine (DPC) identified two amphipathic alpha-helical domains. The first domain, S401-F412, corresponds to the helix 8 common to G protein-coupled receptors while the second domain, A440-M461, is a newly identified structural motif in the distal region of the carboxyl-terminus of the receptor. Molecular modeling of the C-tail in DPC indicates that both helices lie parallel to the plane of the membrane with their hydrophobic and hydrophilic faces poised for critical interactions.

Identification of amino acid residues in BK virus VP1 that are critical for viability and growth.

BK virus (BKV) is a ubiquitous pathogen that establishes a persistent infection in the urinary tract of 80% of the human population. Like other polyomaviruses, the major capsid protein of BKV, virion protein 1 (VP1), is critical for host cell receptor recognition and for proper virion assembly. BKV uses a carbohydrate complex containing alpha(2,3)-linked sialic acid attached to glycoprotein and glycolipid motifs as a cellular receptor. To determine the amino acids important for BKV binding to the sialic acid portion of the complex, we generated a series of 17 point mutations in VP1 and scored them for viral growth. The first set of mutants behaved identically to wild-type virus, suggesting that these amino acids were not critical for virus propagation. Another group of VP1 mutants rendered the virus nonviable. These mutations failed to protect viral DNA from DNase I digestion, indicating a role for these domains in capsid assembly and/or packaging of DNA. A third group of VP1 mutations packaged DNA similarly to the wild type but failed to propagate. The initial burst size of these mutations was similar to that of the wild type, indicating that there is no defect in the lytic release of the mutated virions. Binding experiments revealed that a subset of the BKV mutants were unable to attach to their host cells. These motifs are likely important for sialic acid recognition. We next mapped these mutations onto a model of BKV VP1 to provide atomic insight into the role of these sites in the binding of sialic acid to VP1.

Activation of ERK, JNK, Akt, and G-protein coupled signaling by hybrid angiotensin II AT1/bradykinin B2 receptors expressed in HEK-293 cells.

Bradykinin (BK) and angiotensin II (AngII) often have opposite roles in cardiovascular diseases. Our aim here was to construct hybrid receptors which bind AngII but signal as BK. Various sequences of the intracellular face of the AngII type I receptor, AT1R, were replaced with corresponding sequences from the bradykinin B2 receptor (BKB2R). The hybrids demonstrated a number of signaling characteristics of the BKB2R. For example, the hybrids demonstrated BK as opposed to AngII like phosphorylation of Akt and JNK. The hybrids containing the BKB2R intracellular loop 2 (IC2) displayed minimal G-protein, Galphai/Galphaq, linked signaling. Computer based molecular models suggested that Ser-Met-Gly from the IC2 of the BKB2R is detrimental for the Galphai/Galphaq coupled functions of this hybrid. The return of Lys-Ser-Arg of the AT1R to this hybrid led to almost full recovery of Galphai and Galphaq activation. The design and production of AT1/BKB2 hybrid receptors is a potential approach in the treatment of hypertension related diseases where the presence of AngII, its AT1 receptor and the consequent signal transduction has proven detrimental.

Structural features of parathyroid hormone receptor coupled to Galpha(s)-protein.

The molecular basis of the activation of G-proteins by the G-protein coupled receptor for parathyroid hormone (PTH) is unknown. Employing a combination of NMR methods and computer-based structural refinement, structural features involved in the activation of Galpha(s) by the PTH receptor (PTH1R) have been determined. Focusing on the C-terminus of the third intracellular loop (IC3), previously shown to be important for Galpha(s) activation by PTH1R, the structure of this region, PTH1R(402-408), while bound to Galpha(s), was determined by transferred nuclear Overhauser effect spectroscopy. The relative topological orientation of the IC3 while associated with Galpha(s) was determined by saturation transfer difference spectroscopy. These experimental data were incorporated into molecular dynamics simulations of the PTH1R and Galpha(s) to provide atomic insight into the receptor-protein interactions important for PTH signaling and a structural framework to analyze previous mutagenesis studies of Galpha(s). These data provide the first step toward development of a molecular mechanism for the signaling profile of PTH1R, an important regulator of calcium levels in the bloodstream.

The conformation of cyclo(-D-Pro-Ala4-) as a model for cyclic pentapeptides of the DL4 type.

The conformation of the cyclic pentapeptide cyclo(-D-Pro-Ala(4)-) in solution and in the solid state was reinvestigated using modern NMR techniques. To allow unequivocal characterization of hydrogen bonds, relaxation behavior, and intramolecular distances, differently labeled isotopomers were synthesized. The NMR results, supported by extensive MD simulations, demonstrate unambiguously that the preferred conformation previously described by us, but recently questioned, is indeed correct. The validation of the conformational preferences of this cyclic peptide is important given that this system is a template for several bioactive compounds and for controlled "spatial screening" for the search of bioactive conformations.

The role of sialic acid in human polyomavirus infections.

JC virus (JCV) and BK virus (BKV) are human polyomaviruses that infect approximately 85% of the population worldwide [1,2]. JCV is the underlying cause of the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML), a condition resulting from JCV induced lytic destruction of myelin producing oligodendrocytes in the brain [3]. BKV infection of kidneys in renal transplant recipients results in a gradual loss of graft function known as polyomavirus associated nephropathy (PVN) [4]. Following the identification of these viruses as the etiological agents of disease, there has been greater interest in understanding the basic biology of these human pathogens [5,6]. Recent advances in the field have shown that viral entry of both JCV and BKV is dependent on the ability to interact with sialic acid. This review focuses on what is known about the human polyomaviruses and the role that sialic acid plays in determining viral tropism.

Defining the retinoid binding site in the rod cyclic nucleotide-gated channel.

Rod vision is initiated when 11-cis-retinal, bound within rhodopsin, absorbs a photon and isomerizes to all-trans-retinal (ATR). This triggers an enzyme cascade that lowers cGMP, thereby closing cyclic nucleotide-gated (CNG) channels. ATR then dissociates from rhodopsin, with bright light releasing millimolar levels of ATR. We have recently shown that ATR is a potent closed-state inhibitor of the rod CNG channel, and that it requires access to the cytosolic face of the channel (McCabe, S.L., D.M. Pelosi, M. Tetreault, A. Miri, W. Nguitragool, P. Kovithvathanaphong, R. Mahajan, and A.L. Zimmerman. 2004. J. Gen. Physiol. 123:521-531). However, the details of the interaction between the channel and ATR have not been resolved. Here, we explore the nature of this interaction by taking advantage of specific retinoids and retinoid analogues, namely, beta-ionone, all-trans-C15 aldehyde, all-trans-C17 aldehyde, all-trans-C22 aldehyde, all-trans-retinol, all-trans-retinoic acid, and all-trans-retinylidene-n-butylamine. These retinoids differ in polyene chain length, chemical functionality, and charge. Results obtained from patch clamp and NMR studies have allowed us to better define the characteristics of the site of retinoid-channel interaction. We propose that the cytoplasmic face of the channel contains a retinoid binding site. This binding site likely contains a hydrophobic region that allows the ionone ring and polyene tail to sit in an optimal position to promote interaction of the terminal functional group with residues approximately 15 A away from the ionone ring. Based on our functional data with retinoids possessing either a positive or a negative charge, we speculate that these amino acid residues may be polar and/or aromatic.

Modeling a sialic acid binding pocket in the external loops of JC virus VP1.

JC virus (JCV) is a common human polyomavirus that infects over 70% of the population worldwide. JCV has a restricted cell tropism that is caused partly by the initial interaction between the virus and sialic acid-containing host cell receptors. To identify the molecular interactions between the virus and its cellular receptor, we used a combined approach of site-directed mutagenesis and homology-based molecular modeling. A model of the major viral capsid protein VP1 based on sequence alignment with other closely related polyomaviruses allowed us to target specific amino acids in the extracellular loops of VP1 for mutagenesis. An analysis of the growth rates of 17 point mutants led to the identification of VP1 amino acids that are critical in virus-host cell receptor interactions. Molecular dynamics simulations were then used to build and confirm a model of the interaction between VP1 and the sialic acid component of the JCV receptor.

Cooperative interaction of arginine-19 and the N-terminal signaling domain in the affinity and potency of parathyroid hormone.

Residue 19 of parathyroid hormone (PTH) plays a unique role in the interaction process with the PTH1 receptor. A Glu(19) --> Arg(19) substitution, based on the Arg(19) of the PTH-related protein (PTHrP), increases the binding affinity when incorporated into the N-terminus of PTH [i.e., PTH(1-20)] and has no effect when introduced into the C-terminus of PTH [i.e., PTH(15-31)]. To explore Arg(19) and the midregion (residues 10-15), we designed the novel PTH scaffold peptide, PG5, which has the PTH(1-9) domain linked to the PTH(15-31) segment via a pentaglycine spacer. Substitution of Glu(19) with Arg(19) in PG5 resulted in a 9-fold increase in binding affinity. Additionally, the substitution enhanced stimulated cAMP formation in cells expressing PTH1-delNt, a PTH1 receptor construct lacking most of the N-terminus, confirming that residue 19 is interacting with the juxtamembrane portion of PTH1. The binding and signaling capacities of the PG5 analogues were diminished relative to those of PTH(1-34), indicating that the residue 10-14 region of PTH provides more than just a simple linker function. To probe this further, the structural consequences of the glycine linker and its interaction with PTH1 were examined by circular dichroism, (1)H NMR, and extensive ligand/receptor molecular dynamics simulations. The structural data clearly illustrate the helix-stabilizing effect of Arg(19) substitution propagating N-terminally from position 19 to the pentaglycine linker. Overall, these studies suggest that an alpha-helix is the preferred conformation for the residue 15-20 region of PTH and that residues 10-14 are also required for full affinity and potency of the hormone.

Toward parathyroid hormone minimization: conformational studies of cyclic PTH(1-14) analogues.

The N-terminal fragment of PTH(1-34) is critical for PTH1 receptor activation. Various modifications of PTH(1-14) have been shown to result in a considerable increase in signaling potency [Shimizu et al. (2000) J. Biol. Chem. 275, 21836-21843]. Our structural investigations revealed an unusually stable helical structure of the signaling domain (1-14), where residues 6 (Gln) and 10 (Gln or Asn) were located on the same face of the alpha-helix. To test whether a stable N-terminal alpha-helix is required for productive interaction with PTH1 receptor, we designed two conformationally restricted PTH(1-14) analogues, each containing a lactam bridge at positions 6 and 10. Specifically, substitutions Gln(6)-->Glu(6) and Asn(10)-->Lys(10) were introduced into the most potent [Ala(1,3,12),Gln(10),Har(11),Trp(14)]PTH(1-14)NH2 agonist. Both the Glu(6)-Lys(10) and Lys(6)-Glu(10) lactam-bridged analogues were characterized to examine the importance of orientation of the lactam. According to biological studies [Shimizu et al. (2003) Biochemistry 42, 2282-2290], none of the 6/10 substituted analogues (linear or cyclic) remained as active as the parent peptide. However, relative to their corresponding linear peptides, lactam-bridged analogues either maintained potency or showed 6-fold improvement. High-resolution structures as determined by 1H NMR and NOE-restrained molecular dynamics simulations clearly illustrate the structural differences between the linear and cyclic PTH(1-14) fragments, supporting the hypothesis that an alpha-helix is the preferred bioactive conformation of the N-terminal fragment of PTH. In addition, our results demonstrate that the structural order of the very first residues (1-4) of the signaling domain plays a significant role in PTH action.

19F MAS NMR quantification of accessible hydroxyl sites on fiberglass surfaces.

Solid-state 19F nuclear magnetic resonance (NMR) spectroscopy is used for the quantitative investigation of accessible hydroxyl sites on low surface area glass fibers. Samples with surface areas as low as 0.2 m2/g are investigated through covalent binding of (3,3,3-trifluoropropyl)dimethylchlorosilane. 19F is an ideal nucleus for solid-state NMR, as it has a nuclear spin of 1/2 and a natural isotopic abundance of 100%. High-speed MAS techniques (with rotor spinning frequencies greater than 15 kHz) sufficiently average the CSA and any strong dipolar couplings to allow for superior resolution, especially from terminal -CF3 groups. Studies of two model silica gels with higher surface area, but different pore sizes, provide chemical shift and spin-lattice relaxation rate parameters for probe molecules bound within different environments: pores approaching the size of the probe molecule and pores much larger than the molecular size where intermolecular interactions are assumed to be at a minimum. Resonances assignable to both types of binding environments are found in the spectra of similarly functionalized low surface area fibers. Accessible hydroxyl coverages in the range of 0.8-1.3 OH/nm2 have been measured, and an initial discussion of fiber surface roughness and microporosity is advanced.