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1.
Clin Drug Investig ; 44(8): 587-599, 2024 Aug.
Article in English | MEDLINE | ID: mdl-39073504

ABSTRACT

BACKGROUND AND OBJECTIVES: Nipocalimab is a high-affinity, fully human, effectorless immunoglobulin G1 monoclonal antibody targeting the neonatal Fc receptor and is currently under evaluation for the treatment of rare and prevalent immunoglobulin G autoantibody-mediated and alloantibody-mediated diseases. This phase I, randomized, double-blind, placebo-controlled, single-dose escalation study in healthy Japanese volunteers (N = 24) assessed the safety, pharmacokinetics, and effect on the serum immunoglobulin G level of single doses of nipocalimab. METHODS: Volunteers were grouped into three cohorts and received intravenous nipocalimab at 10, 30, or 60 mg/kg or placebo. To complement the study, genetic variation in the Fcγ receptor and transporter subunit of the neonatal Fc receptor was analyzed in Japanese and diverse populations. RESULTS: Nipocalimab was generally safe and well tolerated at all dose levels, with three (12.5% [3/24]) volunteers experiencing treatment-emergent adverse events across all nipocalimab doses. Mean serum immunoglobulin G levels decreased in a dose-dependent manner from baseline with nipocalimab treatment compared with placebo. Maximum serum nipocalimab concentrations demonstrated proportional increases with dose, while the area under the concentration-time curve was dose dependent and demonstrated non-linear increases with dose. Mean observed half-life was longer as the dose increased. Analysis of genetic variation in Fcγ receptor and transporter identified no unique Japanese variants or variants that alter amino acid sequences in or near the neonatal Fc receptor immunoglobulin G binding site targeted by nipocalimab. CONCLUSIONS: The comparable pharmacokinetic/pharmacodynamic profiles and highly conserved neonatal Fc receptor structure among diverse populations further support the clinical development of nipocalimab for the treatment of various immunoglobulin G autoantibody-mediated and alloantibody-mediated diseases across global sites and populations, including the Japanese population.


Subject(s)
Healthy Volunteers , Immunoglobulin G , Receptors, Fc , Adult , Female , Humans , Male , Middle Aged , Young Adult , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal, Humanized/administration & dosage , Antibodies, Monoclonal, Humanized/pharmacokinetics , Dose-Response Relationship, Drug , Double-Blind Method , East Asian People , Histocompatibility Antigens Class I/genetics , Japan , Receptors, Fc/antagonists & inhibitors
2.
Cell Rep ; 43(3): 113965, 2024 Mar 26.
Article in English | MEDLINE | ID: mdl-38492217

ABSTRACT

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibits stress granule assembly and interacts with G3BP1/2 via an ITFG motif, including residue F17, in the N protein. Prior studies examining the impact of the G3PB1-N interaction on SARS-CoV-2 replication have produced inconsistent findings, and the role of this interaction in pathogenesis is unknown. Here, we use structural and biochemical analyses to define the residues required for G3BP1-N interaction and structure-guided mutagenesis to selectively disrupt this interaction. We find that N-F17A mutation causes highly specific loss of interaction with G3BP1/2. SARS-CoV-2 N-F17A fails to inhibit stress granule assembly in cells, has decreased viral replication, and causes decreased pathology in vivo. Further mechanistic studies indicate that the N-F17-mediated G3BP1-N interaction promotes infection by limiting sequestration of viral genomic RNA (gRNA) into stress granules.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , DNA Helicases/metabolism , RNA Helicases/metabolism , RNA Recognition Motif Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , Virulence , RNA, Guide, CRISPR-Cas Systems , Nucleocapsid Proteins , Virus Replication , RNA, Viral/genetics
3.
J Cell Biol ; 223(3)2024 03 04.
Article in English | MEDLINE | ID: mdl-38284934

ABSTRACT

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the RNA-binding proteins G3BP1/2. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting the co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress and dissolve pre-existing stress granules. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent powerful tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.


Subject(s)
DNA Helicases , RNA Helicases , Stress Granules , DNA Helicases/genetics , Poly-ADP-Ribose Binding Proteins/genetics , RNA Helicases/genetics , RNA Recognition Motif Proteins/genetics
4.
bioRxiv ; 2023 Jun 30.
Article in English | MEDLINE | ID: mdl-37425880

ABSTRACT

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. G3BP1/2 are prominent interactors of the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the functional consequences of the G3BP1-N interaction in the context of viral infection remain unclear. Here we used structural and biochemical analyses to define the residues required for G3BP1-N interaction, followed by structure-guided mutagenesis of G3BP1 and N to selectively and reciprocally disrupt their interaction. We found that mutation of F17 within the N protein led to selective loss of interaction with G3BP1 and consequent failure of the N protein to disrupt stress granule assembly. Introduction of SARS-CoV-2 bearing an F17A mutation resulted in a significant decrease in viral replication and pathogenesis in vivo, indicating that the G3BP1-N interaction promotes infection by suppressing the ability of G3BP1 to form stress granules.

5.
bioRxiv ; 2023 Jun 28.
Article in English | MEDLINE | ID: mdl-37425931

ABSTRACT

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the paralogs G3BP1 and G3BP2. G3BP1/2 proteins bind mRNAs and thereby promote the condensation of mRNPs into stress granules. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, referred to as G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is known to be targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress, and dissolve pre-existing stress granules when added to cells after stress granule formation. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent ideal tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

6.
Arthritis Res Ther ; 21(1): 216, 2019 10 23.
Article in English | MEDLINE | ID: mdl-31647025

ABSTRACT

BACKGROUND: The goal of this study is to use comprehensive molecular profiling to characterize clinical response to anti-TNF therapy in a real-world setting and identify reproducible markers differentiating good responders and non-responders in rheumatoid arthritis (RA). METHODS: Whole-blood mRNA, plasma proteins, and glycopeptides were measured in two cohorts of biologic-naïve RA patients (n = 40 and n = 36) from the Corrona CERTAIN (Comparative Effectiveness Registry to study Therapies for Arthritis and Inflammatory coNditions) registry at baseline and after 3 months of anti-TNF treatment. Response to treatment was categorized by EULAR criteria. A cell type-specific data analysis was conducted to evaluate the involvement of the most common immune cell sub-populations. Findings concordant between the two cohorts were further assessed for reproducibility using selected NCBI-GEO datasets and clinical laboratory measurements available in the CERTAIN database. RESULTS: A treatment-related signature suggesting a reduction in neutrophils, independent of the status of response, was indicated by a high level of correlation (ρ = 0.62; p < 0.01) between the two cohorts. A baseline, response signature of increased innate cell types in responders compared to increased adaptive cell types in non-responders was identified in both cohorts. This result was further assessed by applying the cell type-specific analysis to five other publicly available RA datasets. Evaluation of the neutrophil-to-lymphocyte ratio at baseline in the remaining patients (n = 1962) from the CERTAIN database confirmed the observation (odds ratio of good/moderate response = 1.20 [95% CI = 1.03-1.41, p = 0.02]). CONCLUSION: Differences in innate/adaptive immune cell type composition at baseline may be a major contributor to response to anti-TNF treatment within the first 3 months of therapy.


Subject(s)
Adaptive Immunity/physiology , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/genetics , Gene Expression Profiling/methods , Immunity, Innate/physiology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Adaptive Immunity/drug effects , Adult , Aged , Antirheumatic Agents/pharmacology , Antirheumatic Agents/therapeutic use , Arthritis, Rheumatoid/immunology , Cohort Studies , Female , Humans , Immunity, Innate/drug effects , Male , Middle Aged , Prospective Studies , Treatment Outcome , Tumor Necrosis Factor-alpha/immunology
7.
Circ Genom Precis Med ; 12(4): e002433, 2019 04.
Article in English | MEDLINE | ID: mdl-30844302

ABSTRACT

BACKGROUND: The sequelae of Kawasaki disease (KD) vary widely with the greatest risk for future cardiovascular events among those who develop giant coronary artery aneurysms (CAA). We sought to define the molecular signature associated with different outcomes in pediatric and adult KD patients. METHODS: Molecular profiling was conducted using mass spectrometry-based shotgun proteomics, transcriptomics, and glycomics methods on 8 pediatric KD patients at the acute, subacute, and convalescent time points. Shotgun proteomics was performed on 9 KD adults with giant CAA and matched healthy controls. Plasma calprotectin was measured by ELISA in 28 pediatric KD patients 1 year post-KD, 70 adult KD patients, and 86 healthy adult volunteers. RESULTS: A characteristic molecular profile was seen in pediatric patients during the acute disease, which resolved at the subacute and convalescent periods in patients with no coronary artery sequelae but persisted in 2 patients who developed giant CAA. We, therefore, investigated persistence of inflammation in KD adults with giant CAA by shotgun proteomics that revealed a signature of active inflammation, immune regulation, and cell trafficking. Correlating results obtained using shotgun proteomics in the pediatric and adult KD cohorts identified elevated calprotectin levels in the plasma of patients with CAA. Investigation of expanded pediatric and adult KD cohorts revealed elevated levels of calprotectin in pediatric patients with giant CAA 1 year post-KD and in adult KD patients who developed giant CAA in childhood. CONCLUSIONS: Complex patterns of biomarkers of inflammation and cell trafficking can persist long after the acute phase of KD in patients with giant CAA. Elevated levels of plasma calprotectin months to decades after acute KD and infiltration of cells expressing S100A8 and A9 in vascular tissues suggest ongoing, subclinical inflammation. Calprotectin may serve as a biomarker to inform the management of KD patients following the acute illness.


Subject(s)
Biomarkers/blood , Coronary Aneurysm/diagnosis , Leukocyte L1 Antigen Complex/blood , Mucocutaneous Lymph Node Syndrome/pathology , Acute Disease , Adult , C-Reactive Protein/analysis , Calgranulin A/metabolism , Calgranulin B/metabolism , Case-Control Studies , Child , Coronary Vessels/metabolism , Humans , Inflammation/etiology , Myocardium/metabolism , Phenotype , Proteomics
8.
Mol Cell Proteomics ; 18(3): 534-545, 2019 03.
Article in English | MEDLINE | ID: mdl-30559323

ABSTRACT

The importance of IgG glycosylation, Fc-gamma receptor (FcγR) single nucleotide polymorphisms and FcγR copy number variations in fine tuning the immune response has been well established. There is a growing appreciation of the importance of glycosylation of FcγRs in modulating the FcγR-IgG interaction based on the association between the glycosylation of recombinant FcγRs and the kinetics and affinity of the FcγR-IgG interaction. Although glycosylation of recombinant FcγRs has been recently characterized, limited knowledge exists on the glycosylation of endogenous human FcγRs. In order to improve the structural understanding of FcγRs expressed on human cells we characterized the site specific glycosylation of native human FcγRIII from neutrophils of 50 healthy donors and from matched plasma for 43 of these individuals. Through this analysis we have confirmed site specific glycosylation patterns previously reported for soluble FcγRIII from a single donor, identified FcγRIIIb specific Asn45 glycosylation and an allelic effect on glycosylation at Asn162 of FcγRIIIb. Identification of FcγRIIIb specific glycosylation allows for assignment of FcγRIIIb alleles and relative copy number of the two alleles where DNA/RNA is not available. Intriguingly the types of structures found to be elevated at Asn162 in the NA2 allele have been shown to destabilize the Fc:FcγRIII interaction resulting in a faster dissociation rate. These differences in glycosylation may in part explain the differential activity reported for the two alleles which have similar in vitro affinity for IgG.


Subject(s)
Asparagine/chemistry , Receptors, IgG/chemistry , Receptors, IgG/metabolism , GPI-Linked Proteins/chemistry , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Gene Dosage , Genotype , Glycosylation , Healthy Volunteers , Humans , Immunoglobulin Fc Fragments/metabolism , Mannose/chemistry , Mass Spectrometry , Models, Molecular , Neutrophils/immunology , Plasma/immunology , Receptors, IgG/genetics
9.
Clin Pharmacol Ther ; 105(4): 1031-1039, 2019 04.
Article in English | MEDLINE | ID: mdl-30402880

ABSTRACT

M281 is a fully human, anti-neonatal Fc receptor (FcRn) antibody that inhibits FcRn-mediated immunoglobulin G (IgG) recycling to decrease pathogenic IgG while preserving IgG production. A randomized, double-blind, placebo-controlled, first-in-human study with 50 normal healthy volunteers was designed to probe safety and the physiological maximum for reduction of IgG. Intravenous infusion of single ascending doses up to 60 mg/kg induced dose-dependent serum IgG reductions, which were similar across all IgG subclasses. Multiple weekly doses of 15 or 30 mg/kg achieved mean IgG reductions of ≈85% from baseline and maintained IgG reductions ≥75% from baseline for up to 24 days. M281 was well tolerated, with no serious or severe adverse events (AEs), few moderate AEs, and a low incidence of infection-related AEs similar to placebo treatment. The tolerability and consistency of M281 pharmacokinetics and pharmacodynamics support further evaluation of M281 in diseases mediated by pathogenic IgG.


Subject(s)
Antibodies/metabolism , Antibodies/therapeutic use , Histocompatibility Antigens Class I/metabolism , Immunoglobulin G/metabolism , Receptors, Fc/metabolism , Adult , Antibodies/adverse effects , Double-Blind Method , Female , Healthy Volunteers , Humans , Infusions, Intravenous/methods , Male , Young Adult
10.
CNS Neurol Disord Drug Targets ; 16(6): 714-723, 2017.
Article in English | MEDLINE | ID: mdl-28240190

ABSTRACT

BACKGROUND: In April 2015, the US Food and Drug Administration approved the first generic glatiramer acetate, Glatopa® (M356), as fully substitutable for Copaxone® 20 mg/mL for relapsing forms of multiple sclerosis (MS). This approval was accomplished through an Abbreviated New Drug Application that demonstrated equivalence to Copaxone. METHOD: This article will provide an overview of the methods used to establish the biological and immunological equivalence of the two glatiramer acetate products, including methods evaluating antigenpresenting cell (APC) biology, T-cell biology, and other immunomodulatory effects. RESULTS: In vitro and in vivo experiments from multiple redundant orthogonal assays within four biological processes (aggregate biology, APC biology, T-cell biology, and B-cell biology) modulated by glatiramer acetate in MS established the biological and immunological equivalence of Glatopa and Copaxone and are described. The following were observed when comparing Glatopa and Copaxone in these experiments: equivalent delays in symptom onset and reductions in "disease" intensity in experimental autoimmune encephalomyelitis; equivalent dose-dependent increases in Glatopa- and Copaxone- induced monokine-induced interferon-gamma release from THP-1 cells; a shift to a T helper 2 phenotype resulting in the secretion of interleukin (IL)-4 and downregulation of IL-17 release; no differences in immunogenicity and the presence of equivalent "immunofingerprints" between both versions of glatiramer acetate; and no stimulation of histamine release with either glatiramer acetate in basophilic leukemia 2H3 cell lines. CONCLUSION: In summary, this comprehensive approach across different biological and immunological pathways modulated by glatiramer acetate consistently supported the biological and immunological equivalence of Glatopa and Copaxone.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/drug therapy , Encephalomyelitis, Autoimmune, Experimental/immunology , Glatiramer Acetate/therapeutic use , Immunosuppressive Agents/therapeutic use , Animals , Antigen-Presenting Cells/drug effects , B-Lymphocytes/drug effects , Cytokines/metabolism , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/chemically induced , Histamine/metabolism , Mice , Myelin Proteolipid Protein/toxicity , Myelin-Oligodendrocyte Glycoprotein/toxicity , Peptide Fragments/toxicity , T-Lymphocytes/drug effects , Therapeutic Equivalency
12.
Nat Immunol ; 17(2): 204-13, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26726811

ABSTRACT

Adjuvanted vaccines afford invaluable protection against disease, and the molecular and cellular changes they induce offer direct insight into human immunobiology. Here we show that within 24 h of receiving adjuvanted swine flu vaccine, healthy individuals made expansive, complex molecular and cellular responses that included overt lymphoid as well as myeloid contributions. Unexpectedly, this early response was subtly but significantly different in people older than ∼35 years. Wide-ranging adverse clinical events can seriously confound vaccine adoption, but whether there are immunological correlates of these is unknown. Here we identify a molecular signature of adverse events that was commonly associated with an existing B cell phenotype. Thus immunophenotypic variation among healthy humans may be manifest in complex pathophysiological responses.


Subject(s)
Influenza A Virus, H1N1 Subtype/immunology , Influenza Vaccines/immunology , Influenza, Human/immunology , Influenza, Human/metabolism , Lymphocytes/immunology , Lymphocytes/metabolism , Adjuvants, Immunologic , Adolescent , Adult , Age Factors , Aged , Antibodies, Viral/blood , Antibodies, Viral/immunology , Autoantibodies/blood , Autoantibodies/immunology , Autoimmunity , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cluster Analysis , Cytokines/blood , Cytokines/metabolism , Female , Gene Expression Profiling , Humans , Influenza Vaccines/adverse effects , Influenza, Human/prevention & control , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Lymphocyte Count , Male , Middle Aged , Myeloid Cells/immunology , Myeloid Cells/metabolism , Phenotype , Time Factors , Transcriptome , Vaccination , Young Adult
13.
PLoS One ; 10(10): e0140299, 2015.
Article in English | MEDLINE | ID: mdl-26473741

ABSTRACT

Glatopa™ is a generic glatiramer acetate recently approved for the treatment of patients with relapsing forms of multiple sclerosis. Gene expression profiling was performed as a means to evaluate equivalence of Glatopa and Copaxone®. Microarray analysis containing 39,429 unique probes across the entire genome was performed in murine glatiramer acetate--responsive Th2-polarized T cells, a test system highly relevant to the biology of glatiramer acetate. A closely related but nonequivalent glatiramoid molecule was used as a control to establish assay sensitivity. Multiple probe-level (Student's t-test) and sample-level (principal component analysis, multidimensional scaling, and hierarchical clustering) statistical analyses were utilized to look for differences in gene expression induced by the test articles. The analyses were conducted across all genes measured, as well as across a subset of genes that were shown to be modulated by Copaxone. The following observations were made across multiple statistical analyses: the expression of numerous genes was significantly changed by treatment with Copaxone when compared against media-only control; gene expression profiles induced by Copaxone and Glatopa were not significantly different; and gene expression profiles induced by Copaxone and the nonequivalent glatiramoid were significantly different, underscoring the sensitivity of the test system and the multiple analysis methods. Comparative analysis was also performed on sets of transcripts relevant to T-cell biology and antigen presentation, among others that are known to be modulated by glatiramer acetate. No statistically significant differences were observed between Copaxone and Glatopa in the expression levels (magnitude and direction) of these glatiramer acetate-regulated genes. In conclusion, multiple methods consistently supported equivalent gene expression profiles between Copaxone and Glatopa.


Subject(s)
Gene Expression Profiling , Gene Expression Regulation/drug effects , Glatiramer Acetate/pharmacology , Th2 Cells/immunology , Animals , Female , Gene Expression Regulation/immunology , Mice , Mice, Inbred BALB C , Multiple Sclerosis/drug therapy , Multiple Sclerosis/immunology
14.
J Med Chem ; 57(11): 4511-20, 2014 Jun 12.
Article in English | MEDLINE | ID: mdl-24786387

ABSTRACT

To date, the structure-activity relationship studies of heparin/heparan sulfate with their diverse binding partners such as growth factors, cytokines, chemokines, and extracellular matrix proteins have been limited yet provide early insight that specific sequences contribute to this manifold biological role. This has led to an impetus for the chemical synthesis of oligosaccharide fragments of these complex polysaccharides, which can provide an effective tool for this goal. The synthesis of three heparin mimetic hexasaccharides with distinct structural patterns is described herein, and the influence of the targeted substitution on their bioactivity profiles is studied using in vitro affinity and/or inhibition toward different growth factors and proteins. Additionally, the particularly challenging synthesis of an irregular hexasaccharide is reported, which, interestingly, in spite of being considerably structurally similar with its two counterparts, displayed a unique and remarkably distinct profile in the test assays.


Subject(s)
Heparin/chemistry , Oligosaccharides/chemical synthesis , Cytokines/chemistry , Glucuronidase/antagonists & inhibitors , Intercellular Signaling Peptides and Proteins/chemistry , Molecular Mimicry , Oligosaccharides/chemistry , Protein Binding , Structure-Activity Relationship , Sulfonic Acids/chemical synthesis , Sulfonic Acids/chemistry
15.
Anal Bioanal Chem ; 406(13): 3079-89, 2014 May.
Article in English | MEDLINE | ID: mdl-24664406

ABSTRACT

The binding affinity and specificity of heparin to proteins is widely recognized to be sulfation-pattern dependent. However, for the majority of heparin-binding proteins (HBPs), it still remains unclear what moieties are involved in the specific binding interaction. Here, we report our study using saturation transfer difference (STD) nuclear magnetic resonance (NMR) to map out the interactions of synthetic heparin oligosaccharides with HBPs, such as basic fibroblast growth factor (FGF2) and fibroblast growth factor 10 (FGF10), to provide insight into the critical epitopes of heparin ligands involved. The irradiation frequency of STD NMR was carefully chosen to excite the methylene protons so that enhanced sensitivity was obtained for the heparin-protein complex. We believe this approach opens up additional application avenues to further investigate heparin-protein interactions.


Subject(s)
Fibroblast Growth Factor 10/metabolism , Fibroblast Growth Factor 2/metabolism , Heparin/metabolism , Magnetic Resonance Spectroscopy/methods , Fibroblast Growth Factor 10/chemistry , Fibroblast Growth Factor 2/chemistry , Heparin/chemistry , Humans , Protein Binding , Surface Plasmon Resonance
16.
PLoS One ; 6(6): e21106, 2011.
Article in English | MEDLINE | ID: mdl-21698156

ABSTRACT

Heparan sulfate proteoglycans (HSPGs) play a key role in shaping the tumor microenvironment by presenting growth factors, cytokines, and other soluble factors that are critical for host cell recruitment and activation, as well as promoting tumor progression, metastasis, and survival. M402 is a rationally engineered, non-cytotoxic heparan sulfate (HS) mimetic, designed to inhibit multiple factors implicated in tumor-host cell interactions, including VEGF, FGF2, SDF-1α, P-selectin, and heparanase. A single s.c. dose of M402 effectively inhibited seeding of B16F10 murine melanoma cells to the lung in an experimental metastasis model. Fluorescent-labeled M402 demonstrated selective accumulation in the primary tumor. Immunohistological analyses of the primary tumor revealed a decrease in microvessel density in M402 treated animals, suggesting anti-angiogenesis to be one of the mechanisms involved in-vivo. M402 treatment also normalized circulating levels of myeloid derived suppressor cells in tumor bearing mice. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis and prolonged survival in an orthotopic 4T1 murine mammary carcinoma model. These data demonstrate that modulating HSPG biology represents a novel approach to target multiple factors involved in tumor progression and metastasis.


Subject(s)
Disease Progression , Heparitin Sulfate/analogs & derivatives , Heparitin Sulfate/pharmacology , Melanoma, Experimental/pathology , Molecular Mimicry , Neoplasm Metastasis , Animals , Cell Line, Tumor , Flow Cytometry , Melanoma, Experimental/blood supply , Mice , Surface Plasmon Resonance
17.
Glycobiology ; 21(9): 1194-205, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21515908

ABSTRACT

A series of size-defined low-molecular-weight heparins were generated by regioselective chemical modifications and profiled for their in vitro and in vivo activities. The compounds displayed reduced anti-coagulant activity, demonstrated varying affinities toward angiogenic growth factors (fibroblast growth factor-2, vascular endothelial growth factor and stromal cell-derived factor-1α), inhibited the P-selectin/P-selectin glycoprotein ligand-1 interaction and, notably, exhibited anti-tumor efficacy in a murine melanoma experimental metastasis model. Our results demonstrate that modulating specific sequences, especially the N-domains (-NS or -NH(2) or -NHCOCH(3)) in these polysaccharide sequences, has a major impact on the participation in a diverse range of biological activities. These results also suggest that the 6-O-sulfates, but not the 2-O-sulfates, critically affect the binding of a desulfated derivative to certain angiogenic proteins as well as its ability to inhibit P-selectin-mediated B16F10 melanoma metastases. Furthermore, N-desulfation followed by N-acetylation regenerates the affinity/inhibition properties to different extents in all the compounds tested in the in vitro assays. This systematic study lays a conceptual foundation for detailed structure function elucidation and will facilitate the rational design of targeted heparan sulfate proteoglycan-based anti-metastatic therapeutic candidates.


Subject(s)
Heparin, Low-Molecular-Weight , Lung Neoplasms/drug therapy , Melanoma, Experimental/drug therapy , Neovascularization, Pathologic/drug therapy , Small Molecule Libraries , Animals , Binding Sites , Chemokine CXCL12/antagonists & inhibitors , Chemokine CXCL12/metabolism , Drug Design , Female , Fibroblast Growth Factor 2/antagonists & inhibitors , Fibroblast Growth Factor 2/metabolism , Heparin, Low-Molecular-Weight/chemistry , Heparin, Low-Molecular-Weight/metabolism , Heparin, Low-Molecular-Weight/pharmacology , High-Throughput Screening Assays , Hydrolysis , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Neoplasm Transplantation , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Neovascularization, Pathologic/prevention & control , P-Selectin/antagonists & inhibitors , P-Selectin/metabolism , Protein Binding , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , Structure-Activity Relationship , Sulfates/metabolism , Surface Plasmon Resonance , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/metabolism
18.
Bioconjug Chem ; 21(1): 14-9, 2010 Jan.
Article in English | MEDLINE | ID: mdl-20028085

ABSTRACT

Nanoparticles bearing surface-conjugated targeting ligands are increasingly being explored for a variety of biomedical applications. The multivalent conjugation of targeting ligands on the surface of nanoparticles is presumed to enhance binding to the desired target. However, given the complexities inherent in the interactions of nanoparticle surfaces with proteins, and the structural diversity of nanoparticle scaffolds and targeting ligands, our understanding of how conjugation of targeting ligands affects nanoparticle binding remains incomplete. Here, we use surface plasmon resonance (SPR) to directly and quantitatively study the affinity and binding kinetics of nanoparticles that display small molecules conjugated to their surface. We studied the interaction between a single protein target and a structurally related series of targeting ligands whose intrinsic affinity varies over a 4500-fold range and performed SPR at protein densities that reflect endogenous receptor densities. We report that even weak small molecule targeting ligands can significantly enhance target-specific avidity (by up to 4 orders of magnitude) through multivalent interactions and also observe a much broader range of kinetic effects than has been previously reported. Quantitative measurement of how the affinity and kinetics of nanoparticle binding vary as a function of different surface conjugations is a rapid, generalizable approach to nanoparticle characterization that can inform the design of nanoparticles for biomedical applications.


Subject(s)
Molecular Probe Techniques , Nanoparticles/analysis , Nanoparticles/chemistry , Proteins/metabolism , Kinetics , Ligands , Protein Binding , Proteins/chemistry , Receptors, Cell Surface/metabolism , Surface Plasmon Resonance
19.
J Neurochem ; 110(2): 557-69, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19457112

ABSTRACT

Phosphorylation of neurotransmitter receptors can modify their activity and regulate neuronal excitability. Cyclin-dependent kinase 5 (cdk5) is a proline-directed serine/threonine kinase involved not only in neuronal development, but also in synaptic function and plasticity. Here we demonstrate that group I metabotropic glutamate receptors (mGluRs), which modulate post-synaptic signaling by coupling to intracellular signal transduction pathways, are phosphorylated by cdk5. In vitro kinase assays reveal that cdk5 phosphorylates mGluR5 within the domain of the receptor that interacts with the scaffolding protein homer. Using a novel phosphospecific mGluR antibody, we show that the homer-binding domain of both mGluR1 and mGluR5 are phosphorylated in vivo, and that inhibition of cdk5 with siRNA decreases the amount of phosphorylated receptor. Furthermore, kinetic binding analysis, by surface plasmon resonance, indicates that phosphorylation of mGluR5 enhances its association with homer. Homer protein complexes in the post-synaptic density, and their disruption by an activity-dependent short homer 1a isoform, have been shown to regulate the trafficking and signaling of the mGluRs and impact many neuroadaptive processes. Phosphorylation of the mGluR homer-binding domain, in contrast to homer 1a induction, provides a novel mechanism for potentially regulating a subset of homer interactions.


Subject(s)
Carrier Proteins/metabolism , Cyclin-Dependent Kinase 5/metabolism , Receptors, Metabotropic Glutamate/metabolism , Amino Acid Sequence , Animals , COS Cells , Carrier Proteins/chemistry , Cells, Cultured , Chlorocebus aethiops , Cyclin-Dependent Kinase 5/chemistry , Homer Scaffolding Proteins , Humans , Mice , Mice, Knockout , Molecular Sequence Data , Phosphorylation/physiology , Protein Binding/physiology , Rats , Rats, Sprague-Dawley , Receptors, AMPA/metabolism , Receptors, Kainic Acid/metabolism , Receptors, Metabotropic Glutamate/chemistry
20.
Nat Chem Biol ; 5(3): 154-6, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19151731

ABSTRACT

Small-molecule inhibition of extracellular proteins that activate membrane receptors has proven to be extremely challenging. Diversity-oriented synthesis and small-molecule microarrays enabled the discovery of robotnikinin, a small molecule that binds the extracellular Sonic hedgehog (Shh) protein and blocks Shh signaling in cell lines, human primary keratinocytes and a synthetic model of human skin. Shh pathway activity is rescued by small-molecule agonists of Smoothened, which functions immediately downstream of the Shh receptor Patched.


Subject(s)
Hedgehog Proteins/metabolism , Lactams/pharmacology , Lactones/pharmacology , Signal Transduction/drug effects , 3T3 Cells , Animals , Drug Discovery , Humans , Keratinocytes/drug effects , Keratinocytes/metabolism , Lactams/metabolism , Lactones/metabolism , Mice , Patched Receptors , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism
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