Molecular Docking of Phytochemicals Targeting GFRs as Therapeutic Sites for Cancer: an In Silico Study.

Drug delivery in a safe manner is a major challenge in the drug development process. Growth factor receptors (GFRs) are known to have profound roles in the growth and progression of cancerous cells making these receptors a therapeutic target in the effective treatment of cancer. This work focused on exploring bioactive compounds that can target GFRs using in silico method. In this study, 50 bioactive compounds from different plant sources were screened as anticancer agent against GFRs using drug likeness parameters of Lipinski's rule of five. The molecular docking was performed between phytochemicals and GFRs. Ligands with acceptable drug likeness and binding energy comparable to the standard drugs were further screened to determine their pharmacokinetic activities. This study showed phytochemicals with the binding energy comparable with the standard drugs (Dovitinib and Gefitinib), while ADME, bioactivity score, and bioavailability radar analysis gave further insight on these compounds as potent anticancer agents.

Revising the mechanism of p75NTR activation: intrinsically monomeric state of death domains invokes the "helper" hypothesis.

The neurotrophin receptor p75NTR plays crucial roles in neuron development and regulates important neuronal processes like degeneration, apoptosis and cell survival. At the same time the detailed mechanism of signal transduction is unclear. One of the main hypotheses known as the snail-tong mechanism assumes that in the inactive state, the death domains interact with each other and in response to ligand binding there is a conformational change leading to their exposure. Here, we show that neither rat nor human p75NTR death domains homodimerize in solution. Moreover, there is no interaction between the death domains in a more native context: the dimerization of transmembrane domains in liposomes and the presence of activating mutation in extracellular juxtamembrane region do not lead to intracellular domain interaction. These findings suggest that the activation mechanism of p75NTR should be revised. Thus, we propose a novel model of p75NTR functioning based on interaction with "helper" protein.

Quantitative super-resolution single molecule microscopy dataset of YFP-tagged growth factor receptors.

Background: Super-resolution single molecule localization microscopy (SMLM) is a method for achieving resolution beyond the classical limit in optical microscopes (approx. 200 nm laterally). Yellow fluorescent protein (YFP) has been used for super-resolution single molecule localization microscopy, but less frequently than other fluorescent probes. Working with YFP in SMLM is a challenge because a lower number of photons are emitted per molecule compared with organic dyes, which are more commonly used. Publically available experimental data can facilitate development of new data analysis algorithms. Findings: Four complete, freely available single molecule super-resolution microscopy datasets on YFP-tagged growth factor receptors expressed in a human cell line are presented, including both raw and analyzed data. We report methods for sample preparation, for data acquisition, and for data analysis, as well as examples of the acquired images. We also analyzed the SMLM datasets using a different method: super-resolution optical fluctuation imaging (SOFI). The 2 modes of analysis offer complementary information about the sample. A fifth single molecule super-resolution microscopy dataset acquired with the dye Alexa 532 is included for comparison purposes. Conclusions: This dataset has potential for extensive reuse. Complete raw data from SMLM experiments have typically not been published. The YFP data exhibit low signal-to-noise ratios, making data analysis a challenge. These datasets will be useful to investigators developing their own algorithms for SMLM, SOFI, and related methods. The data will also be useful for researchers investigating growth factor receptors such as ErbB3.

Click Biotinylation of PLGA Template for Biotin Receptor Oriented Delivery of Doxorubicin Hydrochloride in 4T1 Cell-Induced Breast Cancer.

PLGA was functionalized with PEG and biotin using click chemistry to generate a biotin receptor targeted copolymer (biotinylated-PEG-PLGA) which in turn was used to fabricate ultrafine nanoparticles (BPNP) of doxorubicin hydrochloride (DOX) for effective delivery in 4T1 cell induced breast cancer. However, adequate entrapment of a hydrophilic bioactive like DOX in a hydrophobic polymer system made of PLGA is not usually possible. We therefore modified a conventional W/O/W emulsion method by utilizing NH4Cl in the external phase to constrain DOX in dissolved polymer phase by suppressing DOX's inherent aqueous solubility as per common ion effect. This resulted in over 8-fold enhancement in entrapment efficiency of DOX inside BPNP, which otherwise is highly susceptible to leakage due to its relatively high aqueous solubility. TEM and DLS established BPNP to be sized below 100 nm, storage stability studies showed that BPNP were stable for one month at 4 °C, and in vitro release suggested significant control in drug release. Extensive in vitro and in vivo studies were conducted to propound anticancer and antiproliferative activity of BPNP. Plasma and tissue distribution study supplemented by pertinent in vivo fluorescence imaging mapped the exact fate of DOX contained inside BPNP once it was administered intravenously. A comparative safety profile via acute toxicity studies in mice was also generated to out rightly establish usefulness of BPNP. Results suggest that BPNP substantially enhance anticancer activity of DOX while simultaneously mitigating its toxic potential due to altered spatial and temporal presentation of drug and consequently deserve further allometric iteration.

Probing the dynamics of growth factor receptor by single-molecule fluorescence imaging.

Single-molecule fluorescence imaging with living cells offers a new approach to visualize and monitor individual proteins during their cellular activities. It facilities the study of cell signaling proteins whose structures and interactions are highly temporal and spatial regulated. In this review, we will mainly present our recent work on probing the dynamics of two types of important transmembrane growth factor receptors, transforming growth factor ß receptors and epidermal growth factor receptors, by single-molecule fluorescence microscopy. This includes the characterization of receptor stoichiometry, monomer-dimer interconversion kinetics, effects of microenvironment on receptor membrane diffusion, and intracellular transportation under different signaling conditions. Related studies on these receptors from other groups, as well as the method developments, will be also discussed. Single-molecule study helps to achieve a better understanding of the molecular mechanism of receptor activation, endocytosis and other molecular events in transmembrane signaling.

[(Cp-R)M(CO)3] (M = Re or 99mTc) conjugates for theranostic receptor targeting.

Cyclopentadienyl complexes of 99mTc became accessible via a retro Diels-Alder synthetic approach of dimerized cyclopentadiene derivatives. So far, this approach was limited to derivatives comprising a carboxylic acid group, directly conjugated to the Cp-ring, leading to complexes [(C5H5COOH)99mTc(CO)3] and [(C5H5CONH-R)99mTc(CO)3], respectively. The introduction of an -NCO group via Curtius rearrangement and subsequent in situ reactions with alcohols or amines gave [(C5H5NHCO-OR)2] and [(C5H5NHCO-NHR)2]. To increase the spacer lengths between the Cp-ring and the functional groups, methylene and ethylene spacers were introduced to yield C5H5-CH2COOH and C5H5-C2H4COOH respectively. The latter Cp-derivatives reacted with [99mTcO4)]- and in the presence of CO releasing/reducing agents to the corresponding [(C5H5-spacer-COOH)99mTc(CO)3] complexes. The carboxylato groups can be derivatized with targeting functions, leading to structurally altered receptor binding complexes, with 99mTc for imaging and with rhenium for therapy. The nature of the 99mTc complexes was assessed by HPLC comparison with the corresponding rhenium compounds.

Coiled-coil-mediated grafting of bioactive vascular endothelial growth factor.

Chimeric growth factors may represent a powerful alternative to their natural counterparts for the functionalization of tissue-engineered scaffolds and applications in regenerative medicine. Their rational design should provide a simple, readily scalable production strategy while improving retention at the site of action. In that endeavor, we here report the synthesis of a chimeric protein corresponding to human vascular endothelial growth factor 165 being N-terminally fused to an E5 peptide tag (E5-VEGF). E5-VEGF was successfully expressed as a homodimer in mammalian cells. Following affinity purification, in vitro surface plasmon resonance biosensing and cell survival assays confirmed diffusible E5-VEGF ability to bind to its receptor ectodomains, while observed morphological phenotypes confirmed its anti-apoptotic features. Additional surface plasmon resonance assays highlighted that E5-VEGF could be specifically captured with high stability when interacting with covalently immobilized K5 peptide (a synthetic peptide designed to bind to the E5 moiety of chimeric hVEGF). This immobilization strategy was applied to glass substrates and chimeric hVEGF was shown to be maintained in a functionally active state following capture. Altogether, our data demonstrated that stable hVEGF capture can be performed via coiled-coil interactions without impacting hVEGF bioactivity, thus opening up the way to future applications in the field of tissue engineering and regenerative medicine.

Understanding cytokine and growth factor receptor activation mechanisms.

Our understanding of the detailed mechanism of action of cytokine and growth factor receptors - and particularly our quantitative understanding of the link between structure, mechanism and function - lags significantly behind our knowledge of comparable functional protein classes such as enzymes, G protein-coupled receptors, and ion channels. In particular, it remains controversial whether such receptors are activated by a mechanism of ligand-induced oligomerization, versus a mechanism in which the ligand binds to a pre-associated receptor dimer or oligomer that becomes activated through subsequent conformational rearrangement. A major limitation to progress has been the relative paucity of methods for performing quantitative mechanistic experiments on unmodified receptors expressed at endogenous levels on live cells. In this article, we review the current state of knowledge on the activation mechanisms of cytokine and growth factor receptors, critically evaluate the evidence for and against the different proposed mechanisms, and highlight other key questions that remain unanswered. New approaches and techniques have led to rapid recent progress in this area, and the field is poised for major advances in the coming years which promise to revolutionize our understanding of this large and biologically and medically important class of receptors.

One step synthesis of gold-loaded radial mesoporous silica nanospheres and supported lipid bilayer functionalization: towards bio-multifunctional sensors.

A simple synthetic route is developed to achieve gold functionalized radial mesoporous silica nanoparticles (Au-MsNP) synthesized by a one step procedure fully compatible with basic conditions required for the preparation of monodispersed nanospheres. In a second step, Au-MsNP particles have been coated with phospholipid bilayers in order to design an advanced biofunctional platform with the gold metallic nanoparticles previously grown into the pore channels and responsible for a plasmonic activity relevant for biosensing. The size of Au-MsNP is checked by dynamic light scattering while zeta potential measurements reflect their surface charge. The particle morphology is characterized by transmission and scanning electron microscopy and the Si/Au ratios are obtained from energy dispersive X-ray analysis. The textural properties of Au-MsNP, specific surface area and pore size, are determined from N(2) adsorption. The supported bilayers are achieved from vesicles of different phospholipids incubated with Au-MsNP particles. The coating efficiency is investigated by zeta potential and cryo- transmission electron microscopy. The plasmonic activities of bare Au-MsNP particles and coated lipid bilayer Au-MsNP platform are evidenced for two model systems: direct adsorption of bovine serum albumin and molecular recognition events between avidin molecules and biotin receptors integrated in the supported lipid bilayer.

Gold nanoparticles surface-functionalized with paclitaxel drug and biotin receptor as theranostic agents for cancer therapy.

We describe in this study whether the gold nanoparticle (AuNP) surface-functionalized with PEG, biotin, paclitaxel (PTX) and rhodamine B linked beta-cyclodextrin (ß-CD) (AuNP-5') can be useful as a theranostic agent for cancer therapy without the cytotoxic effect on normal cells. Prior to surface-functionalizing AuNPs, the cytotoxicity of the nanoparticles was evaluated, followed by their cytocompatibility. PTX, an anti-cancer agent, formed inclusion complexations with ß-CD conjugated AuNPs, and effectively released from the AuNP-2' surface-functionalized with PEG, beta-cyclodextrin (ß-CD) and paclitaxel (PTX) using the intracellular glutathione (GSH) level (10 mm). Two types of AuNP-4 surface-functionalized with PEG and rhodamine B linked ß-CD and AuNP-5 surface-functionalized PEG, biotin and rhodamine B linked ß-CD were used for evaluating their specific interaction on cancer cells such as HeLa, A549 and MG63. These were also tested against normal NIH3T3 cell, determining that the AuNP-5 was more effectively involved with the cancer cells. Confocal laser scanning microscopy (CLSM), fluorescence-activated cell-sorting (FACS) and cell viability analyses showed that the AuNP-5' plays a significant role in the diagnosis and therapy of the cancer cells, and may be used in theranostic agents.

A drug resistance screen using a selective MET inhibitor reveals a spectrum of mutations that partially overlap with activating mutations found in cancer patients.

The emergence of drug resistance is a primary concern in any cancer treatment, including with targeted kinase inhibitors as exemplified by the appearance of Bcr-Abl point mutations in chronic myeloid leukemia (CML) patients treated with imatinib. In vitro approaches to identify resistance mutations in Bcr-Abl have yielded mutation spectra that faithfully recapitulated clinical observations. To predict resistance mutations in the receptor tyrosine kinase MET that could emerge during inhibitor treatment in patients, we conducted a resistance screen in BaF3 TPR-MET cells using the novel selective MET inhibitor NVP-BVU972. The observed spectrum of mutations in resistant cells was dominated by substitutions of tyrosine 1230 but also included other missense mutations and partially overlapped with activating MET mutations that were previously described in cancer patients. Cocrystallization of the MET kinase domain in complex with NVP-BVU972 revealed a key role for Y1230 in binding of NVP-BVU972, as previously reported for multiple other selective MET inhibitors. A second resistance screen in the same format with the MET inhibitor AMG 458 yielded a distinct spectrum of mutations rich in F1200 alterations, which is consistent with a different predicted binding mode. Our findings suggest that amino acid substitutions in the MET kinase domain of cancer patients need to be carefully monitored before and during treatment with MET inhibitors, as resistance may preexist or emerge. Compounds binding in the same manner as NVP-BVU972 might be particularly susceptible to the development of resistance through mutations in Y1230, a condition that may be addressed by MET inhibitors with alternative binding modes.

Cooperation between integrins and growth factor receptors in signaling and endocytosis.

All multicellular animals express receptors for growth factors (GFs) and extracellular matrix (ECM) molecules. Integrin-type ECM receptors anchor cells to their surroundings and concomitantly activate intracellular signal transduction pathways. The same signaling mechanisms are regulated by GF receptors (GFRs). Recently, intensive research efforts have revealed novel mechanisms describing how the two receptor systems collaborate at many different levels. Integrins can directly bind to GFs and promote their activation. Adhesion receptors also organize signaling platforms and assist GFRs or even activate them via ligand-independent mechanisms. Furthermore, integrins can orchestrate endocytosis and recycling of GFRs. Here, we review the present knowledge about the interplay between integrins and GFRs and discuss recent ideas of how this collaboration may explain some previous controversies in integrin research.

An adjacent arginine, and the phosphorylated tyrosine in the c-Met receptor target sequence, dictates the orientation of c-Cbl binding.

Previously, we have demonstrated that the tyrosine phosphorylated hepatocyte growth factor receptor (Met) binds to the c-Cbl phosphotyrosine-recognition, tyrosine kinase binding (TKB) domain in a reverse orientation compared to other c-Cbl binding partners. A Met peptide with the DpYR motif changed to RpYD (MetRD) retains a similar TKB binding affinity as the native Met peptide. However, the TKB: MetRD complex crystal structure reveals a complete reversal of the binding orientation. Collated data indicates that both binding and orientation is dictated by the phosphorylated tyrosine and an adjacent arginine forming intra-peptide hydrogen bonds and aligning unidirectionally with complementary charges in the phosphotyrosine binding pocket of c-Cbl.

The HGF/MET pathway as target for the treatment of multiple myeloma and B-cell lymphomas.

Hepatocyte growth factor (HGF) and its receptor MET are essential during embryonic development and throughout postnatal life. However, aberrant MET activation, due to overexpression, mutations, or autocrine ligand production, contributes to the development and progression of a variety of human cancers, often being associated with poor clinical outcome and drug resistance. B cell malignancies arise from B cells that are clonally expanded at different stages of differentiation. Despite major therapeutic advances, most mature B cell malignancies remain incurable and biologically-oriented therapeutic strategies are urgently needed. This review addresses the role of the HGF/MET pathway during B cell development and discusses how its aberrant activation contributes to the development of B cell lymphoproliferative disorders, with particular emphasis on multiple myeloma and diffuse large B cell lymphoma. These insights, combined with the recent development of clinical-grade agents targeting the MET pathway, provide the rationale to envision the HGF/MET pathway as a new promising target for the treatment of B cell malignancies.

Cell delivery of Met docking site peptides inhibit angiogenesis and vascular tumor growth.

Hepatocyte growth factor (HGF) and its receptor Met are responsible for a wide variety of cellular responses, both physiologically during embryo development and tissue homeostasis, and pathologically, particularly during tumor growth and dissemination. In cancer, Met can act as an oncogene on tumor cells, as well as a pro-angiogenic factor activating endothelial cells and inducing new vessel formation. Molecules interfering with Met activity could be valuable therapeutic agents. Here we have investigated the antiangiogenic properties of a synthetic peptide mimicking the docking site of the Met carboxyl-terminal tail, which was delivered into the cells by fusion with the internalization sequences from Antennapedia or HIV-Tat. We showed that these peptides inhibit ligand-dependent endothelial cell proliferation, motility, invasiveness and morphogenesis in vitro to an even greater extent and with much less toxicity than the Met inhibitor PHA-665752, which correlated with interference of HGF-dependent downstream signaling. In vivo, the peptides inhibited HGF-induced angiogenesis in the matrigel sponge assay and impaired xenograft tumor growth and vascularization in Kaposi's sarcoma. These data show that interference with the Met receptor intracellular sequence impairs HGF-induced angiogenesis, suggesting the use of antidocking site compounds as a therapeutic strategy to counteract angiogenesis in cancer as well as in other diseases.

Ligand-mediated dimerization of the Met receptor tyrosine kinase by the bacterial invasion protein InlB.

The Listeria monocytogenes surface protein InlB mediates bacterial invasion into host cells by activating the human receptor tyrosine kinase Met. So far, it is unknown how InlB or the physiological Met ligand hepatocyte growth factor/scatter factor causes Met dimerization, which is considered a prerequisite for receptor activation. We determined two new structures of InlB, revealing a recurring, antiparallel, dimeric arrangement, in which the two protomers interact through the convex face of the leucine-rich repeat domain. The same contact is found in one structure of the InlB-Met complex. Mutations disrupting the interprotomeric contact of InlB reduced its ability to activate Met and downstream signaling. Conversely, stabilization of this crystal contact by two intermolecular disulfide bonds generates a constitutively dimeric InlB variant with exceptionally high signaling activity, which can stimulate cell motility and cell division. These data demonstrate that the signaling-competent InlB-Met complex assembles with 2:2 stoichiometry around a back-to-back InlB dimer, enabling the direct contact between the stalk region of two Met molecules.

Tea catechins inhibit hepatocyte growth factor receptor (MET kinase) activity in human colon cancer cells: kinetic and molecular docking studies.

Most cancer deaths result from spread of the primary tumor to distant sites (metastasis). MET is an important protein for metastasis in multiple tumor types. Here we report on the ability of tea catechins to suppress MET activation in human colon cancer cells and propose a mechanism by which they might compete for the kinase domain of the MET protein.

MET pathway as a therapeutic target.

Dysregulation of mesenchymal-epithelial transition factor receptor tyrosine kinase pathway leads to cell proliferation, protection from apoptosis, angiogenesis, invasion, and metastasis. It can be dysregulated through overexpression, constitutive activation, gene amplification, ligand-dependent activation or mutation. New drugs targeting various mesenchymal-epithelial transition factor pathways are being investigated with promising results.

Inhibition of HGF/MET as therapy for malignancy.

BACKGROUND: Inhibition of inappropriate tyrosine kinase activity by neoplasms is an attractive strategy for the treatment of malignancy. OBJECTIVE: We aimed to produce a concise review of the potential role of hepatocyte growth factor (HGF)/Mesenchymal-epithelial transition factor (MET) tyrosine kinase pathway inhibition in the treatment of cancer. METHODS: The current literature, abstracts and internet resources related to HGF/MET structure, function and inhibition are summarized. The potential of inhibiting this pathway as a therapy for cancer and remaining hurdles prior to routine clinical use of MET inhibition are discussed. RESULTS/CONCLUSIONS: Current knowledge suggests that the inhibition of the HGF/MET pathway has significant potential for the treatment of cancer. A number of MET inhibitor molecules are nearing completion of their development for clinical use.

Computational modeling of structurally conserved cancer mutations in the RET and MET kinases: the impact on protein structure, dynamics, and stability.

Structural and biochemical characterization of protein kinases that confer oncogene addiction and harbor a large number of disease-associated mutations, including RET and MET kinases, have provided insights into molecular mechanisms associated with the protein kinase activation in human cancer. In this article, structural modeling, molecular dynamics, and free energy simulations of a structurally conserved mutational hotspot, shared by M918T in RET and M1250T in MET kinases, are undertaken to quantify the molecular mechanism of activation and the functional role of cancer mutations in altering protein kinase structure, dynamics, and stability. The mechanistic basis of the activating RET and MET cancer mutations may be driven by an appreciable free energy destabilization of the inactive kinase state in the mutational forms. According to our results, the locally enhanced mobility of the cancer mutants and a higher conformational entropy are counterbalanced by a larger enthalpy loss and result in the decreased thermodynamic stability. The computed protein stability differences between the wild-type and cancer kinase mutants are consistent with circular dichroism spectroscopy and differential scanning calorimetry experiments. These results support the molecular mechanism of activation, which causes a detrimental imbalance in the dynamic equilibrium shifted toward the active form of the enzyme. Furthermore, computer simulations of the inhibitor binding with the oncogenic and drug-resistant RET mutations have also provided a plausible molecular rationale for the observed differences in the inhibition profiles, which is consistent with the experimental data. Finally, structural mapping of RET and MET cancer mutations and the computed protein stability changes suggest a similar mechanism of activation, whereby the cancer mutations which display the higher oncogenic activity tend to have the greatest destabilization effect on the inactive kinase structure.