Syntheses and activities of new C10 beta-turn peptidomimetics

A program to identify small molecules that mimic or disrupt protein-protein interactions led us to design the peptidomimetics 1-3. Solid-phase syntheses of 1-3 were developed. The purities of the crude materials isolated from the resin tend to be highest for the S- and N-compounds 2 and 3 and better than in the corresponding syntheses of peptidomimetics A. The particular dipeptide units incorporated were chosen to correspond with the turn regions of the neurotrophins (e.g., nerve growth factor [NGF] and the neurotrophin factor-3 [NT-3]). Preliminary studies were performed to access the binding of these analogues to Trk receptors and their ability to induce cell survival (just as NGF and NT-3 do). Several active compounds were identified. However, poor water solubilities of some of the other compounds preclude reliable testing. Consequently, solid-phase modifications to the synthetic procedures were investigated to provide access to the derivatives 12-14 in which the aromatic nitro group is replaced by amine, guanidine, or sulfonamide functionalities. The latter are more acceptable pharmacophores than nitro groups and also tend to increase the water solubilities of the peptidomimetics.     

Influencing receptor-ligand binding mechanisms with multivalent ligand architecture

Multivalent ligands can function as inhibitors or effectors of biological processes. Potent inhibitory activity can arise from the high functional affinities of multivalent ligand-receptor interactions. Effector functions, however, are influenced not only by apparent affinities but also by alternate factors, including the ability of a ligand to cluster receptors. Little is known about the molecular features of a multivalent ligand that determine whether it will function as an inhibitor or effector. We envisioned that, by altering multivalent ligand architecture, ligands with preferences for different binding mechanisms would be generated. To this end, a series of 28 ligands possessing structural diversity was synthesized. This series provides the means to explore the effects of ligand architecture on the inhibition and clustering of a model protein, the lectin concanavalin A (Con A). The structural parameters that were varied include scaffold shape, size, valency, and density of binding elements. We found that ligands with certain architectures are effective inhibitors, but others mediate receptor clustering. Specifically, high molecular weight, polydisperse polyvalent ligands are effective inhibitors of Con A binding, whereas linear oligomeric ligands generated by the ring-opening metathesis polymerization have structural properties that favor clustering. The shape of a multivalent ligand also influences specific aspects of receptor clustering. These include the rate at which the receptor is clustered, the number of receptors in the clusters, and the average interreceptor distance. Our results indicate that the architecture of a multivalent ligand is a key parameter in determining its activity as an inhibitor or effector. Diversity-oriented syntheses of multivalent ligands coupled with effective assays that can be used to compare the contributions of different binding parameters may afford ligands that function by specific mechanisms.     

Role of Nanomechanics in Canonical and Noncanonical Pro-angiogenic Ligand/VEGF Receptor-2 Activation

Vascular endothelial growth factor receptor-2 (VEGFR2) is an endothelial cell receptor that plays a pivotal role in physiologic and pathologic angiogenesis and is a therapeutic target for angiogenesis-dependent diseases, including cancer. By leveraging on a dedicated nanomechanical biosensor, we investigated the nanoscale mechanical phenomena intertwined with VEGFR2 surface recognition by its prototypic ligand VEGF-A and its noncanonical ligand gremlin. We found that the two ligands bind the immobilized extracellular domain of VEGFR2 (sVEGFR2) with comparable binding affinity. Nevertheless, they interact with sVEGFR2 with different binding kinetics and drive different in-plane piconewton intermolecular forces, suggesting that the binding of VEGF-A or gremlin induces different conformational changes in sVEGFR2. These behaviors can be effectively described in terms of a different "nanomechanical affinity" of the two ligands for sVEGFR2, about 16-fold higher for VEGF-A with respect to gremlin. Such nanomechanical differences affect the biological activity driven by the two angiogenic factors in endothelial cells, as evidenced by a more rapid VEGFR2 clustering and a more potent mitogenic response triggered by VEGF-A in respect to gremlin. Together, these data point to surface intermolecular interactions on cell membrane between activated receptors as a key modulator of the intracellular signaling cascade.     

Chemical genetic identification of the histamine H1 receptor as a stimulator of insulin-induced adipogenesis

A large collection of bioactive compounds with diverse biological effects can be used as probes to elucidate new biological mechanisms that influence a particular cellular process. Here we analyze the effects of 880 well-known small-molecule bioactives or drugs on the insulin-induced adipogenesis of 3T3-L1 fibroblasts, a cell-culture model of fat cell differentiation. Our screen identified 86 compounds as modulators of the adipogenic differentiation of 3T3-L1 cells. Examination of their chemical and pharmacological information revealed that antihistamine drugs with distinct chemical scaffolds inhibit differentiation. Histamine H1 receptor is expressed in 3T3-L1 cells, and its knockdown by small interfering RNA impaired the insulin-induced adipogenic differentiation. Histamine receptors and histamine-like biogenic amines may play a role in inducing adipogenesis in response to insulin.     

Singlet oxygen-induced activation of Akt/protein kinase B is independent of growth factor receptors

Singlet oxygen (1O2)-induced cytotoxicity is believed to be responsible for responses to photodynamic therapy and for apoptosis of T helper cells after UV-A treatment. Other cytotoxic oxidants, such as hydrogen peroxide and peroxynitrite have been shown to stimulate cell survival signaling pathways in addition to causing cell death. Both these oxidants stimulate the Akt/protein kinase B survival signaling pathway through activation of membrane tyrosine kinase growth factor receptors. We evaluated the ability of 1O2 to activate the Akt/protein kinase B pathway in NIH 3T3 cells and examined potential activation pathways. Exposure of fibroblasts to 1O2 elicited a strong and sustained phosphorylation of Akt, which occurred concurrently with phosphorylation of p38 kinase, a proapoptotic signal. Inhibition of phosphatidylinositol-3-OH kinase (PI3-K) completely blocked Akt phosphorylation. Significantly, cell death induced by 1O2 was enhanced by inhibition of PI3-K, suggesting that activation of Akt by 1O2 may contribute to fibroblast survival under this form of oxidative stress. 1O2 treatment did not induce phosphorylation of platelet-derived growth factor receptor (PDGFR) or activate SH-PTP2, a substrate of growth factor receptors, suggesting that PDGFR was not activated. In addition, specific inhibition of PDGFR did not affect Akt phosphorylation elicited by 1O2. Activation of neither focal adhesion kinase (FAK) nor Ras protein, both of which mediate responses to reactive oxygen species, appeared to be pathways for the 1O2-induced activation of the PI3-K-Akt survival pathway. Thus, activation of Akt by 1O2 is mediated by PI3-K and contributes to a survival response that counteracts cell death after 1O2-induced injury. However, unlike the response to other oxidants, activation of the PI3-K-Akt by 1O2 does not involve activation of growth factor receptors, FAK or Ras protein.     

Specific interaction between Smad1 and CHIP: a surface plasmon resonance study

The TGF-β superfamily signaling pathway regulates many important biological processes, including cell growth, differentiation and embryonic pattern formation. Smad1, a member of this signaling pathway that functions downstream of serine/threonine kinase receptors, has ability to interact with carboxyl terminus of Hsc70-interacting protein (CHIP), which is an E3 ubiquitin ligase in other cases. It has been reported that Smurf1, a member of the Hect family E3 ubiquitin ligases, can target Smad1 to 26S proteasome for degradation. In this paper, we studied the interaction of Smad1 and CHIP by combination of surface plasmon resonance and supported monolayer approach. The specific binding of Smad1 to CHIP indicates that the degradation of Smad1 may also be mediated by CHIP, and CHIP may play an essential role in the TGF-β signaling pathway.     

Lipopeptides incorporated into supported phospholipid monolayers have high specific activity at low incorporation levels

The ability to present cell adhesion molecule (CAM) ligands in controlled amounts on a culture surface would greatly facilitate the control of cell growth and differentiation. Supported lipid monolayer/bilayer systems have previously been developed that allow for presentation of CAM ligands for cell interaction; however, these systems have employed peptide loadings much higher than those used in poly(ethylene glycol) (PEG)-based immobilization systems. We report the development of synthetic methods that can be used for the efficient and versatile creation of many linear and cyclic lipid-linked peptide moieties. Using RGD-based peptides for the alpha5beta1 integrin as a model system, we have demonstrated that these lipopeptides support efficient cell binding and spreading at CAM ligand loadings as low as 0.1 mol %, which is well below that previously reported for supported lipid systems. Engineered lipopeptide-based surfaces offer unique presentation options not possible with other immobilization systems, and the high activity at low loadings we have shown here may be extremely useful in presenting multiple CAM ligands for studying cell growth, differentiation, and signaling.     

Rewiring cell adhesion

The adhesion of cells is mediated by the binding of several cell-surface receptors to ligands found in the extracellular matrix. These receptors often have overlapping specificities for the peptide ligands, making it difficult to understand the roles for discrete receptors in cell adhesion, migration, and differentiation as well as to direct the selective adhesion of cell types in tissue-engineering applications. To overcome these limitations, we developed a strategy to rewire the receptor-ligand interactions between a cell and substrate to ensure that adhesion is mediated by a single receptor with unique specificity. The strategy combines a genetic approach to engineer the cell surface with a chimeric integrin receptor having a unique ligand binding domain with a surface chemistry approach to prepare substrates that present ligands that are bound by the new binding domain. We show that Chinese hamster ovary cells that are engineered with a chimeric beta1 integrin adhere, signal, and even migrate on a synthetic matrix.     

Synthesis of Indomorphan Pseudo‐Natural Product Inhibitors of Glucose Transporters GLUT‐1 and ‐3

Bioactive compound design based on natural product (NP) structure may be limited because of partial coverage of NP‐like chemical space and biological target space. These limitations can be overcome by combining NP‐centered strategies with fragment‐based compound design through combination of NP‐derived fragments to afford structurally unprecedented “pseudo‐natural products” (pseudo‐NPs). The design, synthesis, and biological evaluation of a collection of indomorphan pseudo‐NPs that combine biosynthetically unrelated indole‐ and morphan‐alkaloid fragments are described. Indomorphane derivative Glupin was identified as a potent inhibitor of glucose uptake by selectively targeting and upregulating glucose transporters GLUT‐1 and GLUT‐3. Glupin suppresses glycolysis, reduces the levels of glucose‐derived metabolites, and attenuates the growth of various cancer cell lines. Our findings underscore the importance of dual GLUT‐1 and GLUT‐3 inhibition to efficiently suppress tumor cell growth and the cellular rescue mechanism, which counteracts glucose scarcity.     

DNA Framework-Based Topological Cell Sorters

Molecular recognition in cell biological process is characterized with specific locks-and-keys interactions between ligands and receptors, which are ubiquitously distributed on cell membrane with topological clustering. Few topologically-engineered ligand systems enable the exploration of the binding strength between ligand-receptor topological organization. Herein, we generate topologically controlled ligands by developing a family of tetrahedral DNA frameworks (TDFs), so the multiple ligands are stoichiometrically and topologically arranged. This topological control of multiple ligands changes the nature of the molecular recognition by inducing the receptor clustering, so the binding strength is significantly improved (ca. 10-fold). The precise engineering of topological complexes formed by the TDFs are readily translated into effective binding control for cell patterning and binding strength control of cells for cell sorting. This work paves the way for the development of versatile design of topological ligands.     

A synthetic mimic of human Fc receptors: defined chemical modification of cell surfaces enables efficient endocytic uptake of human immunoglobulin-G

Binding of ligands to macromolecular receptors on the surface of mammalian cells often results in ligand uptake through receptor-mediated endocytosis. Certain human leukocytes and epithelial cells express Fc receptors (FcRs) that bind and internalize antibodies through this mechanism. To mimic this process, we synthesized an artificial FcR comprising the membrane anchor N-alkyl-3beta-amino-5alpha-cholestane linked to a disulfide-constrained cyclic peptide, termed FcIII, known to exhibit high affinity and specificity for the Fc region of human IgG. Treatment of human Jurkat lymphocytes that lack natural FcRs with the synthetic FcR (1 microM, 1 h) installed an average of approximately 6.2 x 10(5) synthetic receptor molecules per cell surface. These treated cells gained the capacity to internalize human IgG at levels greater than human THP-1 cells that express the natural receptors FcgammaRI and FcgammaRII. By linking binding motifs for circulating ligands to membrane anchors that cycle between the cell surface and intracellular endosomes, minimalistic cell surface receptors can be used to destroy targeted ligands by endocytosis. These small mimics of macromolecular receptors may be useful for controlling the extracellular abundance of ligands involved in disease.     

Nonspanning bivalent ligands as improved surface receptor binding inhibitors of the cholera toxin B pentamer

A series of bivalent ligands of varying length were synthesized to inhibit the receptor-binding process of cholera toxin. Competitive surface receptor binding assays showed that significant potency gains relative to the constituent monovalent ligands were achieved independently from the ability of the extended bivalent ligands to span binding sites within the toxin pentamer. Several models that could account for the unexpected improvement in IC(50) values are examined, taking into account crystallographic analysis of each ligand in complex with the toxin pentamer. Evidence is presented that steric blocking at the receptor binding surface may play a role. The results of our study suggest that the use of relatively short, "nonspanning" bivalent ligands, or monovalent ligands of similar topology and bulk may be an effective way of blocking the interaction of multimeric proteins with their cell surface receptors.     

DNA Framework‐Based Topological Cell Sorters

Molecular recognition in cell biological process is characterized with specific locks‐and‐keys interactions between ligands and receptors, which are ubiquitously distributed on cell membrane with topological clustering. Few topologically‐engineered ligand systems enable the exploration of the binding strength between ligand‐receptor topological organization. Herein, we generate topologically controlled ligands by developing a family of tetrahedral DNA frameworks (TDFs), so the multiple ligands are stoichiometrically and topologically arranged. This topological control of multiple ligands changes the nature of the molecular recognition by inducing the receptor clustering, so the binding strength is significantly improved (ca. 10‐fold). The precise engineering of topological complexes formed by the TDFs are readily translated into effective binding control for cell patterning and binding strength control of cells for cell sorting. This work paves the way for the development of versatile design of topological ligands.     

表皮生长因子受体和抑制剂之间分子对接的研究

研究了表皮生长因子受体(EGFR)和4-苯胺喹唑啉类抑制剂之间的相互作用模式，表皮生长因子受体的三维结构通过同源蛋白模建的方法得到，而抑制剂和靶酶结合复合物结构则通过分子力学和分子动力学结合的方法计算得到。从模拟结果得到的抑制剂和靶酶之间的相互作用模式表明范德华相互作用、疏水相互作用以及氢键相互作用对抑制剂的活性都有重要的影响，抑制剂的苯胺部分位于活性口袋的底部，能够与受体残基的非极性侧链产生很强的范德华和疏水相互作用，抑制剂双环上的取代基团也能和活性口袋外部的部分残基形成一定的范德华和疏水性相互作用，而抑制剂喹唑啉环上的氮原子能和周围的残基形成较强的氢键相互作用，对抑制剂的活性有较大的影响，计算得到抑制剂和靶酶之间的非键相互作用能以及抑制剂和靶酶之间的相互作用信息能够很好地解释抑制剂活性和结构的关系，为全新抑制剂的设计提供了重要的结构信息。     

Small multivalent architectures mimicking homotrimers of the TNF superfamily member CD40L: delineating the relationship between structure and effector function

Synthetic multivalent ligands, owing to the presence of multiple copies of a recognition motif attached to a central scaffold, can mediate clustering of cell surface receptors and thereby function as effector molecules. This paper dissects the relationship between structure and effector function of synthetic multivalent ligands targeting CD40, a cell surface receptor of the tumor necrosis factor receptor (TNF-R) superfamily. Triggering CD40 signaling in vivo can be used to enhance immunity against intracellular pathogens or tumors. A series of multimeric molecules has been prepared by systematically varying the shape and the valency of the central scaffold, the nature and the length of the linker as well as the sequence of the receptor binding motif. The data reported here (i) suggest that radial distribution of CD40-binding units and C3-symmetry are preferred for optimal binding to CD40 and signaling, (ii) underscore the importance of choosing an appropriate linker to connect the receptor binding motif to the central scaffold, and (iii) show the versatility of planar cyclic alpha- and beta-peptides as templates for the design of CD40L mimetics. In particular, the (Ahx)3-B trimeric scaffold-linker combination equally accommodated binding elements derived from distinct CD40L hot-spot regions including AA" loop and beta-strand E. The use of miniCD40Ls such as those reported here is complementary to other approaches (recombinant ligands, agonistic anti-receptor antibodies) and may find interesting therapeutic applications. Furthermore, the results disclosed in this paper provide the basis for future design of other TNF family member mimetics.     

A Bioorthogonal Approach for the Preparation of a Titanium‐Binding Insulin‐like Growth‐Factor‐1 Derivative by Using Tyrosinase

The generation of metal surfaces with biological properties, such as cell‐growth‐enhancing and differentiation‐inducing abilities, could be potentially exciting for the development of functional materials for use in humans, including artificial dental implants and joint replacements. However, currently the immobilization of proteins on the surfaces of the metals are limited. In this study, we have used a mussel‐inspired bioorthogonal approach to design a 3,4‐hydroxyphenalyalanine‐containing recombinant insulin‐like growth‐factor‐1 using a combination of recombinant DNA technology and tyrosinase treatment for the surface modification of titanium. The modified growth factor prepared in this study exhibited strong binding affinity to titanium, and significantly enhanced the growth of NIH3T3 cells on the surface of titanium.     

Neuroprotection signaling pathway of nerve growth factor and brain-derived neurotrophic factor against staurosporine induced apoptosis in hippocampal H19-7 cells

Neurotrophins protect neurons against excitotoxicity; however the signaling mechanisms for this protection remain to be fully elucidated. Here we report that activation of the phosphatidyl inositol 3 kinase (PI3K)/Akt pathway is critical for protection of hippocampal cells from staurosporine (STS) induced apoptosis, characterized by nuclear condensation and activation of the caspase cascade. Both nerve growth factor (NGF) and brain-derived growth factor (BDNF) prevent STS-induced apoptotic morphology and caspase-3 activity by upregulating phosphorylation of the tropomyosin receptor kinase (Trk) receptor. Inhibition of Trk receptor by K252a altered the neuroprotective effect of both NGF and BDNF whereas inhibition of the p75 neurotrophin receptor (p75NTR) had no effect. Impairment of the PI3K/Akt pathway or overexpression of dominant negative (DN)-Akt abolished the protective effect of both neurotrophins, while active Akt prevented cell death. Moreover, knockdown of Akt by si-RNA was able to block the survival effect of both NGF and BDNF. Thus, the survival action of NGF and BDNF against STS-induced neurotoxicity was mediated by the activation of PI3K/Akt signaling through the Trk receptor.     

Monitoring the Reversibility of GPCR Signaling by Combining Photochromic Ligands with Label-free Impedance Analysis

G protein-coupled cell surface receptors (GPCR) trigger complex intracellular signaling cascades upon agonist binding. Classic pharmacological assays provide information about binding affinities, activation or blockade at different stages of the signaling cascade, but real time dynamics and reversibility of these processes remain often disguised. We show that combining photochromic NPY receptor ligands, which can be toggled in their receptor activation ability by irradiation with light of different wavelengths, with whole cell label-free impedance assays allows observing the cell response to receptor activation and its reversibility over time. The concept demonstrated on NPY receptors may be well applicable to many other GPCRs providing a deeper insight into the time course of intracellular signaling processes.