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1.
Front Immunol ; 9: 1558, 2018.
Article in English | MEDLINE | ID: mdl-30050528

ABSTRACT

Epidermolysis bullosa acquisita (EBA) is a rare, but prototypical, organ-specific autoimmune disease, characterized and caused by autoantibodies against type VII collagen (COL7). Mucocutaneous inflammation, blistering, and scarring are the clinical hallmarks of the disease. Treatment of EBA is difficult and mainly relies on general immunosuppression. Hence, novel treatment options are urgently needed. The phosphatidylinositol-3-kinase (PI3K) pathway is a putative target for the treatment of inflammatory diseases, including EBA. We recently discovered LAS191954, an orally available, selective PI3Kδ inhibitor. PI3Kδ has been shown to be involved in B cell and neutrophil cellular functions. Both cell types critically contribute to EBA pathogenesis, rendering LAS191954 a potential drug candidate for EBA treatment. We, here, demonstrate that LAS191954, when administered chronically, dose-dependently improved the clinical phenotype of mice harboring widespread skin lesions secondary to immunization-induced EBA. Direct comparison with high-dose corticosteroid treatment indicated superiority of LAS191954. Interestingly, levels of circulating autoantibodies were unaltered in all groups, indicating a mode of action independent of the inhibition of B cell function. In line with this, LAS191954 also hindered disease progression in antibody transfer-induced EBA, where disease develops dependent on myeloid, but independent of B cells. We further show that, in vitro, LAS191954 dose-dependently impaired activation of human myeloid cells by relevant disease stimuli. Specifically, immune complex-mediated and C5a-mediated ROS release were inhibited in a PI3Kδ-dependent manner. Accordingly, LAS191954 also modulated the dermal-epidermal separation induced in vitro by co-incubation of immune complexes with polymorph nuclear cells, thus pointing to an important role of PI3Kδ in EBA effector functions. Altogether, these results suggest a new potential mechanism for the treatment of EBA and potentially also other autoimmune bullous diseases.

2.
Bioorg Med Chem Lett ; 22(10): 3431-6, 2012 May 15.
Article in English | MEDLINE | ID: mdl-22521646

ABSTRACT

A new class of p38α inhibitors based on a biaryl-triazolopyridine scaffold was investigated. X-ray crystallographic data of the initial lead compound cocrystallised with p38α was crucial in order to uncover a unique binding mode of the inhibitor to the hinge region via a pair of water molecules. Synthesis and SAR was directed towards the improvement of binding affinity, as well as ADME properties for this new class of p38α inhibitors and ultimately afforded compounds showing good in vivo efficacy.


Subject(s)
Benzamides/pharmacology , Mitogen-Activated Protein Kinase 14/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Crystallography, X-Ray , Models, Molecular , Structure-Activity Relationship
3.
Bioorg Med Chem Lett ; 21(21): 6253-7, 2011 Nov 01.
Article in English | MEDLINE | ID: mdl-21958544

ABSTRACT

Crystallographic structural information was used in the design and synthesis of a number of bioisosteric derivatives to replace the amide moiety in a lead series of p38α inhibitors which showed general hydrolytic instability in human liver preparations. Triazole derivative 13 was found to have moderate bioavailability in the rat and demonstrated potent in-vivo activity in an acute model of inflammation.


Subject(s)
Amides/chemistry , Indoles/pharmacology , Protein Kinase Inhibitors/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Hydrogen Bonding , Indoles/chemistry , Indoles/pharmacokinetics , Protein Kinase Inhibitors/chemistry , Rats
4.
J Med Chem ; 54(22): 7899-910, 2011 Nov 24.
Article in English | MEDLINE | ID: mdl-21999461

ABSTRACT

The design, synthesis, and ability to inhibit p38α MAP kinase by a novel series of naphthyridine N-oxides will be described. Some of these compounds showed a significant reduction in the LPS-induced TNFα production in human whole blood. Structure-activity relationship studies revealed that N-oxide oxygen was essential for activity and was probably a determinant factor for its marked selectivity against other related kinases. After an extensive SAR exercise, several compounds from this series were identified as very potent p38α inhibitors. In vivo efficacy of some derivatives was demonstrated to reduce TNFα levels in an acute murine model of inflammation (ED(50) = 0.5 mg/kg in LPS-induced TNFα production when dosed orally 1.5 h prior to LPS administration). The oral efficacy was further demonstrated in a chronic model of adjuvant arthritis in rats with established disease when administered orally (ED(50) < 1 mg/kg).


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Naphthyridines/chemical synthesis , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Arthritis, Experimental/drug therapy , Binding Sites , Cell Line , Crystallography, X-Ray , Humans , Inflammation/drug therapy , Inflammation/etiology , Lipopolysaccharides/pharmacology , Male , Mice , Microsomes, Liver/metabolism , Monocytes/drug effects , Monocytes/metabolism , Naphthyridines/pharmacokinetics , Naphthyridines/pharmacology , Protein Binding , Protein Conformation , Rats , Rats, Wistar , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/biosynthesis
5.
Bioorg Med Chem Lett ; 21(18): 5270-3, 2011 Sep 15.
Article in English | MEDLINE | ID: mdl-21813275

ABSTRACT

Optimisation of a series of indolin-2-one p38α inhibitors was achieved via both blocking of a potential metabolic 'hot spot' and by increasing overall polarity of the lead series leading to non-cytotoxic compounds which showed improved oral bioavailabilities in the rat.


Subject(s)
Indoles/pharmacology , Mitogen-Activated Protein Kinase 14/analysis , Animals , Dose-Response Relationship, Drug , Humans , Indoles/chemical synthesis , Indoles/chemistry , Mitogen-Activated Protein Kinase 14/metabolism , Molecular Structure , Rats , Stereoisomerism , Structure-Activity Relationship
7.
Inflammation ; 34(2): 119-32, 2011 Apr.
Article in English | MEDLINE | ID: mdl-20446028

ABSTRACT

p38 inhibitors are potent TNF-α suppressors in LPS-stimulated human whole blood and promote efficacy in the rat adjuvant arthritis model. However, the anti-TNF-α activity of p38 inhibitors in rat whole blood has not been explored, preventing the establishment of a potential relation between in vitro and in vivo activity data in the same species. We have pharmacologically characterized a rat whole blood assay based on LPS stimulation. While p38 inhibitors showed good activity in the human assay, they failed to inhibit TNF-α in the rat whole blood assay. At high LPS concentration some compounds even potentiated TNF-α production in the rat assay, which could be reverted in the presence of the ERK pathway inhibitor U0126. Our results suggest that p38 contributes directly to TNF-α production in human whole blood while playing a negative regulatory role in rat blood which can be overridden by p38 inhibition in the presence of high stimulus concentration.


Subject(s)
Butadienes/pharmacology , Enzyme Inhibitors/pharmacology , Lipopolysaccharides/immunology , Nitriles/pharmacology , Tumor Necrosis Factor-alpha/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Arthritis, Experimental , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Male , Monocytes/drug effects , Rats , Rats, Wistar , Tumor Necrosis Factor-alpha/blood , p38 Mitogen-Activated Protein Kinases/metabolism
8.
J Med Chem ; 52(17): 5531-45, 2009 Sep 10.
Article in English | MEDLINE | ID: mdl-19678708

ABSTRACT

A novel series of aminopyridine N-oxides were designed, synthesized, and tested for their ability to inhibit p38alpha MAP kinase. Some of these compounds showed a significant reduction in the LPS-induced TNFalpha production in human whole blood. Structure-activity relationship studies revealed that N-oxide oxygen was essential for activity and was probably a determinant factor for a marked selectivity against other related kinases. Compound 45 was identified as a potent and selective p38alpha inhibitor with an appropriate balance between potency and pharmacokinetics. In vivo efficacy of 45 was demonstrated in reducing TNFalpha levels in an acute murine model of inflammation (ED(50) = 1 mg/kg in LPS-induced TNFalpha production when dosed orally 1.5 h prior to LPS administration). The oral efficacy of 45 was further demonstrated in a chronic model of adjuvant arthritis in rats with established disease when administered orally (ED(50) = 4.5 mg/kg).


Subject(s)
Aminopyridines/chemistry , Aminopyridines/pharmacology , Drug Design , Oxides/chemistry , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Aminopyridines/chemical synthesis , Aminopyridines/therapeutic use , Animals , Arthritis, Experimental/blood , Arthritis, Experimental/drug therapy , Catalytic Domain , Cell Line , Humans , Inhibitory Concentration 50 , Male , Models, Molecular , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/therapeutic use , Rats , Rats, Wistar , Structure-Activity Relationship , Substrate Specificity , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/blood , p38 Mitogen-Activated Protein Kinases/chemistry
9.
Drug Discov Today ; 14(19-20): 926-34, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19596080

ABSTRACT

A more complete understanding of the mechanisms that drive autoimmune diseases has begun to be translated into therapeutic options with significant clinical consequences. A clear example of this is the introduction of biological therapies, which have provided new therapeutic avenues, as well as validated the mediators (TNFalpha, IL-6), mechanisms (T cell costimulation, leukocyte migration), and cellular players (T and B lymphocytes) of the disease process itself. New discoveries into the role of Th17 and regulatory T cells and the epigenetic regulation of cytokine expression may offer novel intervention strategies to satisfy the unmet medical needs that still exist in these diseases.


Subject(s)
Autoimmune Diseases/therapy , Autoimmunity/drug effects , Drug Discovery , Genetic Therapy , Immunologic Factors/therapeutic use , Animals , Autoimmune Diseases/genetics , Autoimmune Diseases/immunology , Autoimmunity/genetics , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , Cell Movement/drug effects , Cell Movement/genetics , Cytokines/genetics , Cytokines/metabolism , Epigenesis, Genetic/drug effects , Humans , Signal Transduction/drug effects , Signal Transduction/genetics , T-Lymphocytes/drug effects , T-Lymphocytes/immunology
10.
Mol Immunol ; 45(9): 2446-54, 2008 May.
Article in English | MEDLINE | ID: mdl-18258304

ABSTRACT

TNF is a key factor in a variety of inflammatory diseases. Here we report that TNF induced pro-inflammatory cytokine synthesis of IL-6 and IL-8 is mediated by the Rho GTPase Rac. TNF induces p42/p44, p54 and p38 MAPK kinase; these kinases have been implicated in control of cytokine synthesis. However, over-expression of a dominant negative form of Rac strongly inhibited TNF-induced p42/44 MAPK kinase activation, but had little effect upon JNK and no effect upon p38 MAPK activity. Another key signalling pathway controlling cytokine expression is NF-kappaB. When analyzing TNF-induced NF-kappaB activity via luciferase-reporter assays or via EMSA, we were able to show that the dominant negative version of Rac could completely abrogate TNF-induced NF-kappaB activity. In addition, we also observed that inhibition of the ERK pathway led to a reduction in TNF-induced NF-kappaB transcriptional activity; this was accompanied by an ablation of TNF-induced p65 phosphorylation at serine 276. This would suggest that TNF-induced activation of Rac, lies upstream of NF-kappaB activation, and that the inhibition of this pathway results in inhibition of cytokine production.


Subject(s)
Interleukin-6/biosynthesis , Interleukin-8/biosynthesis , Mitogen-Activated Protein Kinase Kinases/metabolism , NF-kappa B/metabolism , Tumor Necrosis Factor-alpha/metabolism , rac GTP-Binding Proteins/metabolism , Cell Line , Cells, Cultured , Fibroblasts , Humans , Interleukin-6/immunology , Interleukin-8/immunology , NF-kappa B/immunology , Tumor Necrosis Factor-alpha/immunology , rac GTP-Binding Proteins/immunology
11.
FASEB J ; 18(15): 1931-3, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15456737

ABSTRACT

Infection represents a major associated problem in severely burned patients, as it causes skin graft failure and increases the risk of mortality. Topical and systemic antibiotic treatment is limited by the appearance of resistant bacterial strains. Antimicrobial peptides (AMPs) are gene-encoded "natural antibiotics" that form part of the innate mechanism of defense and may be active against such antibiotic-resistant microorganisms. Several microbicidal peptides are expressed in human skin under inflammatory conditions, and their function is not only limited to microbial killing but also influences tissue repair and adaptive immunity. Protein delivery through cutaneous gene therapy is a promising therapeutic tool for both skin and nonskin diseases. Here we present a gene transfer approach aimed at delivering antimicrobial peptides from keratinocytes. Adenoviral vectors encoding antimicrobial peptide genes were used to infect human keratinocytes growing either on plastic or as part of cultured skin equivalents. Inhibition of bacterial growth occurred both in conditioned media and in direct contact with AMPs gene-transduced keratinocytes. In addition, we showed cooperative effects after transfer of combinations of genes encoding for AMPs with structural differences. Combined cutaneous tissue engineering in conjunction with (microbicidal) gene therapy emerges as a tailored therapeutic approach that is useful for wound coverage and, in this case, concomitantly combating infection.


Subject(s)
Anti-Bacterial Agents , Antimicrobial Cationic Peptides/genetics , Genetic Therapy/methods , Keratinocytes/metabolism , Skin Diseases, Bacterial/therapy , Adenoviridae/genetics , Anti-Bacterial Agents/biosynthesis , Antimicrobial Cationic Peptides/biosynthesis , Cell Line , Gene Expression , Genetic Vectors , Humans , In Vitro Techniques , Skin/anatomy & histology , Skin/microbiology , Transduction, Genetic
12.
Oncogene ; 23(10): 1821-8, 2004 Mar 11.
Article in English | MEDLINE | ID: mdl-15014451

ABSTRACT

Malignant gliomas are the most frequently occurring primary brain tumors and are resistant to conventional therapy. Conditionally replicating adenoviruses are a novel strategy in glioma treatment. Clinical trials using E1B mutant adenoviruses have been reported recently and E1A mutant replication-competent adenoviruses are in advanced preclinical testing. Here we constructed a novel replication-selective adenovirus (CB1) incorporating a double deletion of a 24 bp Rb-binding region in the E1a gene, and a 903 bp deleted region in the E1b gene that abrogates the expression of a p53-binding E1B-55 kDa protein. CB1 exerted a potent anticancer effect in vitro in U-251 MG, U-373 MG, and D-54 MG human glioma cell lines, as assessed by qualitative and quantitative viability assays. Replication analyses demonstrated that CB1 replicates in vitro in human glioma cells. Importantly, CB1 acquired a highly attenuated replicative phenotype in both serum-starved and proliferating normal human astrocytes. In vivo experiments using intracranially implanted D-54 MG glioma xenografts in nude mice showed that a single dose of CB1 (1.5 x 10(8) PFU/tumor) significantly improved survival. Immunohistochemical analyses of expressed adenoviral proteins confirmed adenoviral replication within the tumors. The CB1 oncolytic adenovirus induces a potent antiglioma effect and could ultimately demonstrate clinical relevance and therapeutic utility.


Subject(s)
Adenovirus E1A Proteins/genetics , Adenovirus E1B Proteins/genetics , Adenoviruses, Human/genetics , Glioma/pathology , Glioma/virology , Animals , Base Sequence , Cell Line, Tumor , DNA Primers , Gene Deletion , Humans , Polymerase Chain Reaction , Restriction Mapping , Transplantation, Heterologous
13.
J Virol ; 77(11): 6533-40, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12743310

ABSTRACT

Human oncolytic adenoviruses have been used in clinical trials targeting cancers of epithelial origin. To gain a better understanding of the infectious cycle of adenovirus in normal human squamous tissues, we examined the viral infection process in organotypic cultures of primary human keratinocytes. We show that for the infection to occur, wounding of the epithelium is required. In addition, infection appears to initiate at the basal or parabasal cells that express the high-affinity coxsackievirus-adenovirus receptor, CAR, whereas the productive phase takes place in differentiated cells. This is due, at least in part, to the differentiation-dependent activation of the E1A and E2A early promoters and E4 promoters. We also show that adenovirus infection triggers a response mediated by the abnormal accumulation of cyclin E and p21cip1 proteins similar to the one previously observed in human papillomavirus-infected tissues. However, the virus seems to be able to overcome it, at least partially.


Subject(s)
Adenoviridae/pathogenicity , Adenovirus Early Proteins/metabolism , Gene Expression Regulation, Viral , Keratinocytes/virology , Virus Replication , Adenoviridae/physiology , Adenovirus Early Proteins/genetics , Cell Differentiation , Cells, Cultured , Cyclin E/metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/metabolism , Epithelium/injuries , Humans , Keratinocytes/cytology , Promoter Regions, Genetic
14.
Cancer Res ; 62(16): 4663-70, 2002 Aug 15.
Article in English | MEDLINE | ID: mdl-12183423

ABSTRACT

Malignant melanoma is characterized by growing incidence, early metastasis, and a lack of effective treatment for advanced disease, suggesting a pressing need for novel therapeutic approaches. Conditionally replicative adenoviruses (CRAds) constitute a new and promising strategy for cancer treatment that has been rapidly translated into clinical trials. We engineered novel melanoma-targeted CRAds, AdTyrdelta24 and AdTyrdelta2delta24, by replacing the adenoviral E1A promoter with a cassette containing a polyA sequence and a human tyrosinase enhancer/promoter construct (hTyr2E/P). The small size of this cassette allows retention of the E3 region within these CRAds, which was shown to enhance viral spread and oncolysis. In addition, we introduced mutations (delta24 and delta2delta24) into the viral E1A gene, which attenuate adenoviral replication in quiescent cells. The cell cycle pathways mediating this attenuation are defective in melanoma cells. By analysis of E1A expression, we prove fidelity of hTyr2E/P in the adenoviral genome and in the context of viral replication when an upstream polyA was included. We further show efficient cytotoxicity of AdTyrdelta24 and AdTyrdelta2delta24 in melanoma cell lines and a 100-1000-fold attenuation in cell lines derived from various nonmelanocytic tissues. Virus replication and progeny production of these viruses were similarly selective, resulting in 200-800-fold higher virus yields in melanoma cells versus control cells, thus establishing viral cytolysis and spread as the cause of the observed cell killing. Cytotoxicity of AdTyrdelta24 for normal fibroblasts and keratinocytes was strongly attenuated, and this virus caused selective killing of melanoma cells but not surrounding keratinocytes in a coculture system. Progeny production and cytotoxicity of AdTyrdelta24 in melanoma cells were similar to matching viruses containing the stronger cytomegalovirus enhancer/promoter instead of hTyr2E/P. Furthermore, AdTyrdelta24 showed a cytopathic effect similar to the wild-type E1A containing AdTyrwt and only minimally reduced compared with wild-type adenovirus. We conclude that the generated CRAds AdTyrdelta24 and AdTyrdelta2delta24 constitute novel targeted agents for gene therapy and viral oncolysis of metastatic melanoma.


Subject(s)
Adenovirus E1A Proteins/genetics , Adenoviruses, Human/physiology , Melanoma/therapy , Monophenol Monooxygenase/genetics , Adenovirus E1A Proteins/metabolism , Adenoviruses, Human/genetics , Coculture Techniques , Cytopathogenic Effect, Viral , Humans , Keratinocytes/cytology , Keratinocytes/virology , Melanoma/virology , Promoter Regions, Genetic , Retinoblastoma Protein/metabolism , Tumor Cells, Cultured , Virus Replication
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