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1.
Sci Rep ; 11(1): 2118, 2021 01 22.
Article in English | MEDLINE | ID: mdl-33483531

ABSTRACT

Lung fibrosis, or the scarring of the lung, is a devastating disease with huge unmet medical need. There are limited treatment options and its prognosis is worse than most types of cancer. We previously discovered that MK-0429 is an equipotent pan-inhibitor of αv integrins that reduces proteinuria and kidney fibrosis in a preclinical model. In the present study, we further demonstrated that MK-0429 significantly inhibits fibrosis progression in a bleomycin-induced lung injury model. In search of newer integrin inhibitors for fibrosis, we characterized monoclonal antibodies discovered using Adimab's yeast display platform. We identified several potent neutralizing integrin antibodies with unique human and mouse cross-reactivity. Among these, Ab-31 blocked the binding of multiple αv integrins to their ligands with IC50s comparable to those of MK-0429. Furthermore, both MK-0429 and Ab-31 suppressed integrin-mediated cell adhesion and latent TGFß activation. In IPF patient lung fibroblasts, TGFß treatment induced profound αSMA expression in phenotypic imaging assays and Ab-31 demonstrated potent in vitro activity at inhibiting αSMA expression, suggesting that the integrin antibody is able to modulate TGFß action though mechanisms beyond the inhibition of latent TGFß activation. Together, our results highlight the potential to develop newer integrin therapeutics for the treatment of fibrotic lung diseases.


Subject(s)
Antibodies/metabolism , Fibroblasts/metabolism , Integrin alphaV/metabolism , Pulmonary Fibrosis/metabolism , Animals , Antibodies/immunology , Bleomycin , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , Fibroblasts/cytology , Humans , Integrin alphaV/immunology , Male , Mice, Inbred C57BL , Naphthyridines/pharmacology , Propionates/pharmacology , Protein Binding , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/prevention & control
2.
Sci Rep ; 9(1): 16947, 2019 11 18.
Article in English | MEDLINE | ID: mdl-31740740

ABSTRACT

The over-expression and aggregation of α-synuclein (αSyn) are linked to the onset and pathology of Parkinson's disease. Native monomeric αSyn exists in an intrinsically disordered ensemble of interconverting conformations, which has made its therapeutic targeting by small molecules highly challenging. Nonetheless, here we successfully target the monomeric structural ensemble of αSyn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes. Using a surface plasmon resonance high-throughput screen, in which monomeric αSyn is incubated with microchips arrayed with tethered compounds, we identified novel αSyn interacting drug-like compounds. Because these small molecules could impact a variety of αSyn forms present in the ensemble, we tested representative hits for impact on multiple αSyn malfunctions in vitro and in cells including aggregation and perturbation of vesicular dynamics. We thereby identified a compound that inhibits αSyn misfolding and is neuroprotective, multiple compounds that restore phagocytosis impaired by αSyn overexpression, and a compound blocking cellular transmission of αSyn. Our studies demonstrate that drug-like small molecules that interact with native αSyn can impact a variety of its pathological processes. Thus, targeting the intrinsically disordered ensemble of αSyn offers a unique approach to the development of small molecule research tools and therapeutics for Parkinson's disease.


Subject(s)
Small Molecule Libraries/pharmacology , alpha-Synuclein/metabolism , Amyloid/antagonists & inhibitors , Amyloid/metabolism , Cell Line , Fluorescence Resonance Energy Transfer , High-Throughput Screening Assays/methods , Humans , Intrinsically Disordered Proteins/metabolism , Phagocytosis/drug effects , Protein Folding , Small Molecule Libraries/chemistry , Small Molecule Libraries/toxicity , Surface Plasmon Resonance , alpha-Synuclein/chemistry , alpha-Synuclein/drug effects
3.
PLoS One ; 8(8): e71634, 2013.
Article in English | MEDLINE | ID: mdl-24058406

ABSTRACT

Alpha-synuclein protein is strongly implicated in the pathogenesis Parkinson's disease. Increased expression of α-synuclein due to genetic multiplication or point mutations leads to early onset disease. While α-synuclein is known to modulate membrane vesicle dynamics, it is not clear if this activity is involved in the pathogenic process or if measurable physiological effects of α-synuclein over-expression or mutation exist in vivo. Macrophages and microglia isolated from BAC α-synuclein transgenic mice, which overexpress α-synuclein under regulation of its own promoter, express α-synuclein and exhibit impaired cytokine release and phagocytosis. These processes were affected in vivo as well, both in peritoneal macrophages and microglia in the CNS. Extending these findings to humans, we found similar results with monocytes and fibroblasts isolated from idiopathic or familial Parkinson's disease patients compared to age-matched controls. In summary, this paper provides 1) a new animal model to measure α-synuclein dysfunction; 2) a cellular system to measure synchronized mobilization of α-synuclein and its functional interactions; 3) observations regarding a potential role for innate immune cell function in the development and progression of Parkinson's disease and other human synucleinopathies; 4) putative peripheral biomarkers to study and track these processes in human subjects. While altered neuronal function is a primary issue in PD, the widespread consequence of abnormal α-synuclein expression in other cell types, including immune cells, could play an important role in the neurodegenerative progression of PD and other synucleinopathies. Moreover, increased α-synuclein and altered phagocytosis may provide a useful biomarker for human PD.


Subject(s)
Immunity, Innate , Parkinson Disease/diagnosis , Parkinson Disease/immunology , alpha-Synuclein/immunology , Aged , Aged, 80 and over , Animals , Cells, Cultured , Cytokines/immunology , Female , Fibroblasts/immunology , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Macrophages/immunology , Macrophages/metabolism , Macrophages/pathology , Male , Mice , Mice, Transgenic , Microglia/immunology , Microglia/metabolism , Microglia/pathology , Middle Aged , Monocytes/immunology , Monocytes/metabolism , Monocytes/pathology , Parkinson Disease/genetics , Parkinson Disease/pathology , Phagocytosis , Up-Regulation , alpha-Synuclein/genetics
5.
Bioorg Med Chem Lett ; 23(14): 4117-9, 2013 Jul 15.
Article in English | MEDLINE | ID: mdl-23743283

ABSTRACT

Mitsunobu reactions were employed to link t-butyl esters of α4 integrin inhibitors at each of the termini of a three-arm, 40 kDa, branched PEG. Cleavage of the t-butyl esters using HCO2H provided easily isolated PEG derivatives, which are potent α4 integrin inhibitors, and which achieve sustained levels and bioactivity in vivo, following subcutaneous administration to rats.


Subject(s)
Integrin alpha4/chemistry , Polyethylene Glycols/chemistry , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacokinetics , Esters , Half-Life , Humans , Injections, Subcutaneous , Integrin alpha4/immunology , Integrin alpha4/metabolism , Jurkat Cells , Rats
6.
J Immunol ; 182(10): 5909-13, 2009 May 15.
Article in English | MEDLINE | ID: mdl-19414741

ABSTRACT

The humanized anti-alpha(4) integrin Ab Natalizumab is an effective treatment for relapsing-remitting multiple sclerosis. Natalizumab is thought to exert its therapeutic efficacy by blocking the alpha(4) integrin-mediated binding of circulating immune cells to the blood-brain barrier (BBB). As alpha(4) integrins control other immunological processes, natalizumab may, however, execute its beneficial effects elsewhere. By means of intravital microscopy we demonstrate that natalizumab specifically inhibits the firm adhesion but not the rolling or capture of human T cells on the inflamed BBB in mice with acute experimental autoimmune encephalomyelitis (EAE). The efficiency of natalizumab to block T cell adhesion to the inflamed BBB was found to be more effective in EAE than in acute systemic TNF-alpha-induced inflammation. Our data demonstrate that alpha(4) integrin-mediated adhesion of human T cells to the inflamed BBB during EAE is efficiently blocked by natalizumab and thus provide the first direct in vivo proof of concept of this therapy in multiple sclerosis.


Subject(s)
Antibodies, Monoclonal/pharmacology , Blood-Brain Barrier/metabolism , Cell Adhesion/drug effects , Encephalomyelitis, Autoimmune, Experimental/drug therapy , T-Lymphocytes/drug effects , Animals , Antibodies, Monoclonal, Humanized , Chemotaxis, Leukocyte/drug effects , Disease Models, Animal , Female , Humans , Jurkat Cells , Leukocyte Rolling/drug effects , Mice , Microscopy, Fluorescence , Multiple Sclerosis/drug therapy , Natalizumab
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