Inhibition of tumor necrosis factor mRNA translation by a rationally designed immunomodulatory peptide

Based on sequences of immunomodulatory peptides derived from the heavy chain of HLA Class I, novel immunomodulatory peptides with increased potency were developed by computer-aided rational design. Allotrap 1258 was characterized in detail and shown to inhibit cell-mediated immune responses in vitro and in vivo. Immunomodulatory activity was associated with the capability of the peptides to modulate heme oxygenase (HO) activity. In this study we analyzed the effect of Allotrap 1258 on cytokine expression. Allotrap 1258 inhibited concanavalin A- and lipopolysaccharide-induced human and mouse tumor necrosis factor (TNF) production in vitro and in vivo but had no effect on interleukin (IL)-1, IL-2, IL-4, IL-6, or IL-10 expression. Experiments with HO-1/KO and iNOS/KO mice showed that Allotrap 1258-mediated inhibition of TNF was independent of HO-1 and iNOS. Quantitation of TNF protein expression and mRNA steady state levels demonstrated that Allotrap 1258-mediated inhibition occurred at the translational level. Deletion of the AU-rich element in the 3'-untranslated region (UTR) of TNF mRNA, a region known to be involved in TNF mRNA translation, had minimal effect on Allotrap 1258-mediated inhibition. However, replacement of the TNF 3'-UTR with the human globin 3'-UTR rendered the peptide inactive. This demonstrates that besides AU-rich elements, other sequences in the 3'-UTR of TNF mRNA are involved in translational control of TNF expression. Such sequences are necessary for Allotrap 1258-mediated inhibition of TNF production.     

Solution conformation of a rationally designed nonapeptide

Partial 'turn-helix' type modules comprised of LD and DL chiral beta-turns serving as potential helix nucleators have been connected with a view to designing a nascent 'helix-turn-helix' type structure. Conformation of the resultant peptide Boc-(D)Glu-Ala-Aib-Lys-Val-Pro-(D)Asp-Leu-Leu-NHMe has been described in both DMSO and water.     

Ligand-dependent tRNA processing by a rationally designed RNase P riboswitch

We describe a synthetic riboswitch element that implements a regulatory principle which directly addresses an essential tRNA maturation step. Constructed using a rational in silico design approach, this riboswitch regulates RNase P-catalyzed tRNA 5′-processing by either sequestering or exposing the single-stranded 5′-leader region of the tRNA precursor in response to a ligand. A single base pair in the 5′-leader defines the regulatory potential of the riboswitch both in vitro and in vivo. Our data provide proof for prior postulates on the importance of the structure of the leader region for tRNA maturation. We demonstrate that computational predictions of ligand-dependent structural rearrangements can address individual maturation steps of stable non-coding RNAs, thus making them amenable as promising target for regulatory devices that can be used as functional building blocks in synthetic biology.     

Inhibition studies with rationally designed inhibitors of the human low molecular weight protein tyrosine phosphatase

The human low molecular weight protein tyrosine phosphatase (HCPTP) is ubiquitously expressed as two isoforms in a wide range of human cells and may be involved in regulating the metastatic nature of epithelial tumors. A homology model is presented for the HCPTP-B isoform based on known X-ray crystal structures of other low molecular weight PTPs. A comparison of the two isoform structures indicates the possibility of developing isoform-specific inhibitors of HCPTP. Molecular dynamics simulations with CHARMM have been used to study the binding modes of the known adenine effector and phosphate in the active site of both isoforms. This analysis led to the design of the initial lead compound, based on an azaindole ring moiety, which was then also evaluated computationally. A comparison of these simulations indicates the need for a phosphonate group on the indole and provides insight into inhibitor binding modes. Compounds with varying degrees of structural similarity to the azaindole have been synthesized and tested for inhibition with each isoform. These molecular systems were examined with the program AutoDock, and comparisons made with the kinetics and the explicit simulations to validate AutoDock as a screening tool for potential inhibitors. Two compounds were experimentally found to have sub-millimolar inhibition, but the greater solubility of one reinforces the need for experimental testing alongside computational analysis.     

Apolipoprotein E-low density lipoprotein receptor binding: study of protein-protein interaction in rationally selected docked complexes

Apolipoprotein E (apoE) is an important protein involved in lipid metabolism due to its interaction with members of the low-density lipoprotein receptor (LDLR) family. To further understand the molecular basis for this receptor-binding activity, an apoE fragment containing the receptor binding region (residues 135-151) was docked onto the fifth LDLR ligand binding repeat (LR5) by computational methods. A subset of structures generated by the docking was rationally selected on the grounds of experimental data combined with modeling and was used for further analysis. The application and comparison of two different experimental structures for the apoE fragment underlines the local structural changes occurring in apoE when switching from a receptor-inactive to a receptor-active conformation. The body of interactions occurring at the interface between the two proteins is in very good agreement with the biochemical data available for both apoE and LDLR. Charged residues are involved in numerous ionic interactions and might therefore be important for the specificity of the interaction between apoE and LR5. In addition, the interface also features a tryptophan and a stacking of histidine residues, revealing that the association between the two proteins is not entirely governed by ionic interactions. In particular, the presence of histidine residues in the interface gives a structural basis for the pH-regulated release mechanism of apoE in the endosomes. The proposed molecular basis for apoE binding to LDLR could aid the design of strategies for targeting alterations in lipid transport and metabolism.     

A conformationally constrained peptidomimetic binds to the extracellular region of HER2 protein

Human epidermal growth factor receptor 2 (HER2) is a member of the human epidermal growth factor receptor kinases (other members include EGFR or HER1, HER3, and HER4) that are involved in signaling cascades for cell growth and differentiation. It is well established that HER2-mediated heterodimerization has important implications in cancer. Deregulation of signaling pathways and overexpression of HER2 is known to occur in cancer cells, indicating a role of HER2 in tumorigenesis. Therefore, blocking HER2-mediated signaling has potential therapeutic value. We have designed several peptidomimetics to inhibit HER2-mediated signaling for cell growth. One of the compounds (HERP5, Arg-beta Naph-Phe) exhibited antiproliferative activity with IC(50) values in the micromolar-to-nanomolar range in breast cancer cell lines. Binding of fluorescently labeled HERP5 to HER2 protein was evaluated by fluorescence assay, microscopy, and circular dichroism spectroscopy. Results indicated that HERP5 binds to the extracellular region of the HER2 protein. Structure of the peptidomimetic HERP5 was studied by NMR and molecular dynamics simulations. Based on these results a model was proposed for HER2-EGFR dimerization and possible blocking by HERP5 peptidomimetic using a protein-protein docking method.     

Prevention of Alzheimer's disease-associated Abeta aggregation by rationally designed nonpeptidic beta-sheet ligands

A new concept is introduced for the rational design of beta-sheet ligands, which prevent protein aggregation. Oligomeric acylated aminopyrazoles with a donor-acceptor-donor (DAD) hydrogen bond pattern complementary to that of a beta-sheet efficiently block the solvent-exposed beta-sheet portions in Abeta-(1-40) and thereby prevent formation of insoluble protein aggregates. Density gradient centrifugation revealed that in the initial phase, the size of Abeta aggregates was efficiently kept between the trimeric and 15-meric state, whereas after 5 days an additional high molecular weight fraction appeared. With fluorescence correlation spectroscopy (FCS) exactly those two, i.e. a dimeric aminopyrazole with an oxalyl spacer and a trimeric head-to-tail connected aminopyrazole, of nine similar aminopyrazole ligands were identified as efficient aggregation retardants whose minimum energy conformations showed a perfect complementarity to a beta-sheet. The concentration dependence of the inhibitory effect of a trimeric aminopyrazole derivative allowed an estimation of the dissociation constant in the range of 10(-5) m. Finally, electrospray ionization mass spectrometry (ESI-MS) was used to determine the aggregation kinetics of Abeta-(1-40) in the absence and in the presence of the ligands. From the comparable decrease in Abeta monomer concentration, we conclude that these beta-sheet ligands do not prevent the initial oligomerization of monomeric Abeta but rather block further aggregation of spontaneously formed small oligomers. Together with the results from density gradient centrifugation and fluorescence correlation spectroscopy it is now possible to restrict the approximate size of soluble Abeta aggregates formed in the presence of both inhibitors from 3- to 15-mers.     

Study of inhibition effect of Herceptin on interaction between Heregulin and ErbB Receptors HER3/HER2 by single-molecule force spectroscopy

Herceptin is a monoclonal antibody against HER2, which is a member of the epidermal growth factor receptor (ErbB) family and is overexpressed in many cancers. In this work, we have applied single-molecule force spectroscopy to study the effect of Herceptin on HER2 modulated ligand–receptor interaction for ErbB signaling in living cells. Heregulin β1 (HRG), the specific ligand of HER3, was used for HER2 activation as HER3 is the preferable dimerization partner of HER2 and HER3/HER2 is the most representative heterodimer found in cancer. Our results demonstrated a more stable binding of HRG to the cells co-expressing HER3 and HER2 than those expressing HER3 alone. Moreover, the binding force of Herceptin and HER2 is as strong as that of HRG and HER3/HER2. With the addition of Herceptin, the binding strength of HRG to the cells co-expressing HER3 and HER2 decreased. The presence of Herceptin changed the dynamic force spectrum of HRG-HER3/HER2 to that similar to HRG-HER3. Therefore, the enhancement in HRG-HER3 binding after recruiting HER2 was inhibited by Herceptin. The method offers a new approach to study the molecular mechanism of Herceptin anti-cancer effect.     

The interaction between pancreatic lipase and colipase: a protein-protein interaction regulated by a lipid

The eukaryotic transposable element Ty1 is present in about 20-30 integrated copies per yeast aploid genome, variably localized in different strains. Here, we report the presence in yeast of circular extrachromosomal molecules homologous to Ty1, 6 kilobases in size (the same as integrated copies) present in about 1 circular copy/250-300 cells. This finding shows another analogy between eukaryotic-transposable elements and the pro-viral integrative form of retroviruses.     

A rationally designed anticancer drug targeting a unique binding cavity of tubulin

A novel mono-THF containing synthetic anticancer drug (WHI-261) was designed for targeting a previously unrecognized unique narrow binding cavity on the surface of tubulin. The anti-cancer activity of WHI-261 was confirmed using MTT assays. The structure-based design, synthesis, and biological activity of WHI-261 are reported.     

Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2- 3Galbeta1-3GalNAc sequences

The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewisx(Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcN Ac). Much effort has gone into the synthesis of sialylated and sulfated Lewisxanalogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewisxas part of a branched Core 2 O-linked structure, we recently synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(SE-3Galbeta1++ +-3)GalNAc1alphaOMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al. , Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al. , Biochemistry, 31, 9126-9131, 1992; Imai et al. , Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Acalpha2-3Galbeta1-3GalNAc might have the capability of interacting with L- and P-selectin. To examine this hypothesis, we synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Neu5Acalpha2++ +-3Galbeta1-3)- GalNAc alpha1-OB, which was found to be 2- to 3-fold better than sialyl Lexfor P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAcbeta1-4(Fucalpha1- 3)GlcNAcbeta1-OMe (GalNAc- Lewisx-O-methyl glycoside), which also proved to be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5- to 6-fold better at inhibiting L- and P-selectin than sialyl Lewisx-OMe, By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary (beta1-3) arm, and a modified Lexunit on the branched (beta1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLexfor both L- and P-selectin.      

Extension of a de novo TIM barrel with a rationally designed secondary structure element

The ability to construct novel enzymes is a major aim in de novo protein design. A popular enzyme fold for design attempts is the TIM barrel. This fold is a common topology for enzymes and can harbor many diverse reactions. The recent de novo design of a four‐fold symmetric TIM barrel provides a well understood minimal scaffold for potential enzyme designs. Here we explore opportunities to extend and diversify this scaffold by adding a short de novo helix on top of the barrel. Due to the size of the protein, we developed a design pipeline based on computational ab initio folding that solves a less complex sub‐problem focused around the helix and its vicinity and adapt it to the entire protein. We provide biochemical characterization and a high‐resolution X‐ray structure for one variant and compare it to our design model. The successful extension of this robust TIM‐barrel scaffold opens opportunities to diversify it towards more pocket like arrangements and as such can be considered a building block for future design of binding or catalytic sites.     

The Atx1-Ccc2 complex is a metal-mediated protein-protein interaction

Cellular systems allow transition-metal ions to reach or leave the cell or intracellular locations through metal transfer between proteins. By coupling mutagenesis and advanced NMR experiments, we structurally characterized the adduct between the copper chaperone Atx1 and the first copper(I)-binding domain of the Ccc2 ATPase. Copper was required for the interaction. This study provides an understanding of metal-mediated protein-protein interactions in which the metal ion is essential for the weak, reversible interaction between the partners.     

Selection,purification, and characterization of a HER2-targeting soluble designed ankyrin repeat protein by E. coli surface display using HER2-positive melanoma cells

Human epidermal growth factor receptor 2 (HER2) is a powerful target for cancer immune therapy. The development of anti-HER2 monoclonal antibodies targeting different domains of HER2 is quite effective. However, the selection and production of multivalent antibodies are complicated. In this study, a mimivirus-based designed ankyrin repeat protein (DARPin) targeting HER2 was selected from an artificial library by bacteria surface display. The selection was performed on HER2-positive B16BL6/E2 melanoma cells and HER2-nagative cells. DARPin selected from the library could be expressed in soluble form with a yield of 70?mg/L. After purified by two continuous and easy steps, the purity of DARPin was 90% as established by SDS-PAGE and RP-HPLC. Selected DARPin showed significant HER2-targeting ability with an affinity of 1.05?±?0.47?µM. MTT assay demonstrated that at the concentration of 640?nM, the selected DARPin dimer could inhibit the SK-BR-3 growth at a rate of 36.63 and 46.34% in 48 and 72?hr incubation separately, which was similar to trastuzumab (43.12 and 49.14% separately). These findings suggested that it was an effective method to select antibody mimetic DARPin by bacteria surface display combined with live cells sorting and provided a drug candidate for cancer therapy.     

Inhibition of the severe acute respiratory syndrome 3CL protease by peptidomimetic alpha,beta-unsaturated esters

The proteolytic processing of polyproteins by the 3CL protease of severe acute respiratory syndrome coronavirus is essential for the viral propagation. A series of tripeptide alpha,beta-unsaturated esters and ketomethylene isosteres, including AG7088, are synthesized and assayed to target the 3CL protease. Though AG7088 is inactive (IC50 > 100 microM), the ketomethylene isosteres and tripeptide alpha,beta-unsaturated esters containing both P1 and P2 phenylalanine residues show modest inhibitory activity (IC50 = 11-39 microM). The Phe-Phe dipeptide inhibitors 18a-e are designed on the basis of computer modeling of the enzyme-inhibitor complex. The most potent inhibitor 18c with an inhibition constant of 0.52 microM is obtained by condensation of the Phe-Phe dipeptide alpha,beta-unsaturated ester with 4-(dimethylamino)cinnamic acid. The cell-based assays also indicate that 18c is a nontoxic anti-SARS agent with an EC50 value of 0.18 microM.     

TRPM4b channel suppresses store-operated Ca2+ entry by a novel protein-protein interaction with the TRPC3 channel

We identified human TRPC3 protein by yeast two-hybrid screening of a human brain cDNA library with human TRPM4b as a bait. Immunoprecipitation and confocal microscopic analyses confirmed the protein-protein interaction between TRPM4b and TRPC3, and these two TRPs were found to be highly colocalized at the plasma membrane of HEK293T cells. Overexpression of TRPM4b suppressed TRPC3-mediated whole cell currents by more than 90% compared to those in TRPC3-expressed HEK293T cells. Furthermore, HEK293T cells stably overexpressing red fluorescent protein (RFP)-TRPM4b exhibited an almost complete abolition of UTP-induced store-operated Ca²⁺ entry, which is known to take place via endogenous TRPC channels in HEK293T cells. This study is believed to provide the first clear evidence that TRPM4b interacts physically with TRPC3, a member of a different TRP subfamily, and regulates negatively the channel activity, in turn suppressing store-operated Ca²⁺ entry through the TRPC3 channel.     

Direct measurement of protein dynamics inside cells using a rationally designed photoconvertible protein

All biological reactions depend on the diffusion and re-localization of biomolecules. Our understanding of biological processes requires accurate measurement of biomolecule mobility in living cells. Currently, approaches for investigating the mobility of biomolecules are generally restricted to measuring either fast or slow diffusion kinetics. We describe the development and application of a photoconvertible fluorescent protein, Phamret, that can be highlighted by UV light stimulation inducing a change in fluorescence emission from cyan fluorescent protein (CFP) to photoactivated GFP (PA-GFP). Phamret can be monitored by single excitation-dual emission mode for visualization of molecular dynamics for a broad range of kinetics. We also devised a microscopy-based method to measure the diffusion coefficient from the fluorescence decay after photostimulation of Phamret, enabling analysis of diffusion kinetics ranging from less than 0.1 microm2/s up to approximately 100 microm2/s, and found significant changes in free protein movement during cell-cycle progression.     

HER2 targeted molecular MR imaging using a de novo designed protein contrast agent

The application of magnetic resonance imaging (MRI) to non-invasively assess disease biomarkers has been hampered by the lack of desired contrast agents with high relaxivity, targeting capability, and optimized pharmacokinetics. We have developed a novel MR imaging probe targeting to HER2, a biomarker for various cancer types and a drug target for anti-cancer therapies. This multimodal HER20targeted MR imaging probe integrates a de novo designed protein contrast agent with a high affinity HER2 affibody and a near IR fluorescent dye. Our probe can differentially monitor tumors with different expression levels of HER2 in both human cell lines and xenograft mice models. In addition to its 100-fold higher dose efficiency compared to clinically approved non-targeting contrast agent DTPA, our developed agent also exhibits advantages in crossing the endothelial boundary, tissue distribution, and tumor tissue retention over reported contrast agents as demonstrated by even distribution of the imaging probe across the entire tumor mass. This contrast agent will provide a powerful tool for quantitative assessment of molecular markers, and improved resolution for diagnosis, prognosis and drug discovery.