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Psoriasis, psoriatic arthritis, and axial spondyloarthritis are systemic inflammatory diseases, each commonly manifesting as a spectrum of symptoms, complications, and comorbidities that arise differently in individual patients. Drugs targeting inflammatory cytokines common to the pathogenesis of each of these conditions have been developed, although their specific actions in the different tissues involved are variable. For a drug to be effective, it must be efficiently delivered to and locally bioactive in disease-relevant tissues. Detailed clinical data shed light on the therapeutic effects of individual biologics on specific domains or clinical manifestations of disease and assist in guiding treatment decisions. Pharmacologic, molecular, and functional properties of drugs strongly impact their observed safety and efficacy, and an understanding of these properties provides complementary insight. Secukinumab, a fully human monoclonal IgG1/κ antibody selectively targeting interleukin (IL)-17A, has been in clinical use for >6 years in the treatment of moderate to severe psoriasis, psoriatic arthritis, and both radiographic (also known as ankylosing spondylitis) and nonradiographic axial spondyloarthritis. In this review, we discuss pharmacokinetic and pharmacodynamic data for secukinumab to introduce clinicians to the pharmacological properties of this widely used drug. Understanding how these properties affect the observed clinical efficacy, safety, and tolerability of this drug in the treatment of IL-17A-mediated systemic inflammatory diseases is important for all physicians treating these conditions.

[Image: see text] MAP-activated protein kinase 2 (MK2) plays an important role in the regulation of innate immune response as well as in cell survival upon DNA damage. Despite its potential for the treatment of inflammation and cancer, to date no MK2 low molecular weight inhibitors have reached the clinic, mainly due to inadequate absorption, distribution, metabolism, and excretion (ADME) properties. We describe here an approach based on specifically placed fluorine within a recently described pyrrole-based MK2 inhibitor scaffold for manipulation of its physicochemical and ADME properties. While preserving target potency, the novel fluoro-derivatives showed greatly improved permeability as well as enhanced solubility and reduced in vivo clearance leading to significantly increased oral exposure.

BACKGROUND: IκBζ plays a key role in psoriasis by mediating IL-17A-driven effects, but the molecular mechanism by which IL-17A regulates IκBζ expression is not clarified.OBJECTIVE: We sought to explore the molecular transformation in patients with psoriasis during anti-IL-17A (secukinumab) treatment with a focus on IκBζ.METHODS: The study was an open-label, single-arm, single-center secukinumab treatment study that included 14 patients with plaque psoriasis. Skin biopsy specimens and blood samples were collected on days 0, 4, 14, 42, and 84 and processed for microarray gene expression analysis. Furthermore, in vitro experiments with human keratinocytes and synovial fibroblasts were conducted.RESULTS: Secukinumab improved clinical scores and histologic psoriasis features. Moreover, secukinumab altered the skin transcriptome. The major transcriptional shift appeared between day 14 and day 42 after treatment initiation, although 80 genes were differentially expressed already at day 4. Expression of nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor (IκB) ζ (NFKBIZ, the gene encoding IκBζ) was reduced already after 4 days of treatment in the skin. NFKBIZ expression correlated to Psoriasis Area and Severity Index score, and NFKBIZ mRNA levels in the skin decreased during anti-IL-17A treatment. Moreover, specific NFKBIZ signature genes were significantly altered during anti-IL-17A treatment. Finally, we identified NF-κB activator 1 (Act1), p38 mitogen-activated protein kinase (MAPK), Jun NH2-terminal kinase (JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) as key signaling pathways in NFKBIZ/IκBζ regulation.CONCLUSION: Our results define a crucial role for IκBζ in the antipsoriatic effect of secukinumab. Because IκBζ signature genes were regulated already after 4 days of treatment, this strongly indicates that IκBζ plays a crucial role in the antipsoriatic effects mediated by anti-IL-17A treatment.

Multiple sclerosis (MS) is an immune mediated demyelinating disease of the central nervous system (CNS). The importance of immune cells to MS pathology is supported by clinical data linking the depletion of T and B cells, or the prevention of their migration into the brain with significant reduction in relapses and development of new lesions. In vitro studies, preclinical animal models and encouraging data with the anti-IL-17A antibody secukinumab in a small proof of concept study in man, indicate that IL-17A, a key interleukin associated with many inflammatory and autoimmune diseases, may be involved in MS. Not only cells involved in adaptive immune responses such as Th17 cells and cytotoxic T cells, or innate immune responses such as mucosa-associated invariant T (MAIT) cells and γδT cells, but also CNS resident cells such as astrocytes and oligodendrocytes might contribute to the local production of IL-17A. IL-17A synergizes with other proinflammatory cytokines, by inducing the release of additional cytokines, mediators of tissue damage and chemokines, that recruit new inflammatory cells. IL-17A adversely affects the functions of microglia, astrocytes, oligodendrocytes, neurons, neural precursor cells and endothelial cells. Blockade of IL-17A might be beneficial to MS patients not only by inhibiting inflammation and tissue destruction, but also by enhancing repair processes.

Sarcoidosis is a disease characterised by granuloma formation. There is an unmet need for new treatment strategies beyond corticosteroids. The NLRP3 inflammasome pathway is expressed in innate immune cells and senses danger signals to elicit inflammatory interleukin (IL)-1β; it has recently become a druggable target. This prompted us to test the role of the NLRP3 inflammasome and IL-1β pathway in granuloma formation and sarcoidosis.19 sarcoid patients and 19 healthy volunteers were recruited into this pilot study. NLRP3 inflammasome activity was measured in bronchoalveolar lavage (BAL) cells and lung and skin biopsies using immunohistochemistry, Western blot, reverse-transcriptase PCR and ELISA. For in vivo experiments we used the trehalose 6,6′-dimycolate-granuloma mouse model and evaluated lung granuloma burden in miR-223 knockout and NLRP3 knockout mice, as well as the treatment effects of MCC950 and anti-IL-1β antibody therapy.We found strong upregulation of the NLRP3 inflammasome pathway, evidenced by expression of activated NLRP3 inflammasome components, including cleaved caspase-1 and IL-1β in lung granuloma, and increased IL-1β release of BAL cells from sarcoid patients compared to healthy volunteers (p=0.006). mRNA levels of miR-223, a micro-RNA downregulating NLRP3, were decreased and NLRP3 mRNA correspondingly increased in alveolar macrophages from sarcoid patients (pIn conclusion, our data provide evidence of upregulated inflammasome and IL-1β pathway activation in sarcoidosis and suggest both as valid therapeutic targets.

Pyrrolo[2,3-f]isoquinoline based amino acids, tetracyclic lactams and cyclic ketone analogues are described as novel MK2 inhibitors with IC(50) as low as 5nM and good selectivity profiles against a number of related kinases including ERK, p38alpha and JNKs. TNFalpha release was suppressed from human peripheral blood mononuclear cells (hPBMCs), and a representative compound inhibited LPS induced TNFalpha release in mice illustrating the potential of this series to provide orally active MK2 inhibitors.

New, selective 3-aminopyrazole based MK2-inhibitors were discovered by scaffold hopping strategy. The new derivatives proved to inhibit intracellular phosphorylation of hsp27 as well as LPS-induced TNFalpha release in cells. In addition, selected derivative 14e also inhibited LPS-induced TNFalpha release in vivo.

The p38 pathway has been at the center of interest for anti-inflammatory drug discovery for many years as it is crucial for the biosynthesis of TNF-α, IL-1β and other mediators. Most of the anti-inflammatory effects of p38 inhibition are mediated through MAPK-activated protein kinase-2 (MK2), a direct downstream target of p38, which makes MK2 a very interesting drug target. Within the last 5 years, several classes of low-molecular-weight MK2 inhibitors were disclosed in the patent and primary literature. Advanced compounds could be optimized to nanomolar potencies and inhibit TNF-α release, as well as the phosphorylation of the MK2 substrate heat-shock protein 27 in cellular assays. This article will review the recent progress in this field and will highlight and discuss the most promising compound series disclosed so far.

The therapeutic effects of a drug are the result of its action on a specific molecular target combined with the counterregulatory responses of the organism that aim to restore the affected parameters to previous values. The regulatory systems that control the steady state have evolved under natural selection, but because natural selection is a slow process and cannot foresee future developments, these systems are not always adjusted optimally to current conditions. Their activation can therefore reduce or prevent the desired effects of a drug and can even produce adverse effects. Recent examples from the treatment of obesity and arterial hypertension provide new insight to the contribution of such ancient counterregulatory responses to the therapeutic effects of modern pharmacotherapy. This widens the scope of the rapidly growing field of evolutionary medicine, because it demonstrates that evolutionary principles apply not only to the pathogenesis of human diseases, but also to their treatment.

The search for anti-obesity agents has become one of the most exciting areas in drug discovery. Subsequent to an enormous increase in the number of possible molecular targets, the focus has shifted from target identification to target validation. Because important biological functions such as the regulation of energy intake and expenditure are controlled by complex systems, an improved understanding of pathophysiology is a prerequisite for the selection of successful development candidates for the treatment of obesity. Although most of the information on the regulation of energy balance has been obtained from rodents, various monogenic forms of human obesity provide clinical proof of concept for some of these mechanisms. However, it is still not known which are the most promising clinical approaches to lowering body weight and subsequently reducing morbidity and mortality.

The PPP1R3 gene encoding the G-subunit of protein phosphatase-1 has three polymorphisms in linkage disequilibrium in the Pima Indians: an mRNA-destabilizing element in the 3'-untranslated region (ARE1/ARE2 alleles), Arg883Ser, and Asp905Tyr substitutions. The ARE2 allele, Arg883, and Asp905 variants are associated with insulin resistance and higher prevalence of type 2 diabetes in the Pima Indians. The ARE2 allele is associated with lower PPP1R3 transcript and protein levels in muscle tissue. Here we determined the functional contribution of the amino acid substitutions independent of the ARE alleles to insulin-stimulated glycogen synthesis by adenoviral-mediated gene expression in L6 myotubes. Similar overexpression levels of the G-subunit variants increased glycogen synthase fractional activity in the presence ( approximately 1. 5-fold) of insulin compared to control myotubes transduced with adenovirus encoding beta-galactosidase. The glycogen synthesis rate of myotubes overexpressing the G-subunit variants also increased by approximately 1.7-fold over the control with and without insulin. However, these measures were not significantly different among the variants. This study does not support a role for Arg883 and Asp905 variants independent of the ARE2 allele in the impaired insulin-stimulated glycogen synthesis in the muscle of Pima Indians.

The role of the actin cytoskeleton and/or GTPases of the Rho/Rac-family in glucose transport regulation was investigated in 3T3-L1 cells with clostridial toxins which depolymerize actin by inactivation of Rho/Rac (Clostridium difficile toxin B and Clostiridium sordellii lethal toxin (LT)) or by direct ADP-ribosylation (Clostridium botulinum C2 toxin). Toxin B and C2 reduced insulin-stimulated, but not basal, 2-deoxyglucose (2-DOG) uptake rates in 3T3-L1 fibroblasts. In parallel, the toxins produced morphological alterations of the cells reflecting disruption of the actin cytoskeleton. Both toxins reduced the maximum response to insulin but failed to alter the half-maximally stimulating concentrations of insulin. In 3T3-L1 adipocytes, the lethal toxin reduced the effect of insulin on 2-DOG uptake, whereas toxin B and C2 failed to affect glucose transport or cell morphology. When cells were exposed to the toxins after treatment with insulin, both toxin B and the lethal toxin, in contrast to the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin, failed to reduce the 2-DOG uptake rates. Thus, both translocation to the plasma membrane and internalization of glucose transporters were inhibited by the toxins, whereas the PI 3-kinase inhibitor selectively affects translocation. The data suggest that the effects of the clostridial toxins on trafficking of glucose transporters are mediated by the depolymerization of the actin cytoskeleton and are an indirect consequence of Rho or Rac inactivation. It is suggested that pathways signalling through Rac or Rho may play a modulatory role in glucose transport regulation through their effects on the actin network.

The effects of insulin, insulin-like growth factor (IGF)-I, platelet-derived growth factor (PDGF), interleukin (IL)-6 and interferon-γ on 2-deoxyglucose uptake and insulin receptor substrate (IRS)-1 phosphorylation were compared in 3T3-L1 cells at confluence and after differentiation to the adipocyte-like phenotype. Insulin and IGF-I produced the expected stimulation of glucose transport and tyrosine phosphorylation of IRS-1 in both confluent and differentiated cells. In contrast, IL-6 and interferon-γ failed to stimulate glucose transport or IRS-1 phosphorylation, although a marked stimulation of the JAK/STAT pathways as shown by acute-phase response factor (APRF)/Stat3 or Stat1 activation was observed in fibroblasts (IL-6, interferon-γ) and adipocytes (IL-6). PDGF-AA and PDGF-BB stimulated glucose transport in confluent, undifferentiated cells to the same extent as insulin (approximately six-fold stimulation), but produced only a small portion of the effect of insulin in differentiated cells. Similarly, mRNA levels and autophosphorylation of PDGF receptors were much lower in differentiated cells than in confluent fibroblasts. In contrast to insulin and IGF-I, PDGF failed to stimulate tyrosine phosphorylation of IRS-1. All effects of insulin, IGF-I, and PDGF on glucose transport were inhibited by Wortmannin; the half-maximally inhibiting concentration (IC50) of Wortmannin was increased by insulin. These data demonstrate distinct signalling potentials of the investigated receptors, and indicate that the IL-6 and interferon-γ controlled JAK/STAT pathways lack the potential to stimulate glucose transport. IRS-1 does not appear to be involved in the PDGF receptor-mediated effects, whereas activation of phosphatidylinositol (PI) 3-kinase is a crucial event in all pathways leading to stimulation of glucose transport. [Diabetologia (1996) 39: 1432–1439]

The abundance and the subcellular distribution of GTP-binding proteins was studied in membrane fractions (plasma membranes and low-density microsomes) from 3T3-L1 cells before and after differentiation to the insulin-sensitive phenotype. After differentiation, the abundance of alpha i (alpha subunit of GTP-binding protein Gi), alpha o (alpha subunit of GTP-binding protein G(o)), and of a 47-kDa alpha s (alpha subunit of GTP-binding protein Gs) as detected by immunoblotting with specific antisera was reduced by 10-50% when normalized per membrane protein. In contrast, a 43-kDa alpha s was increased about threefold after differentiation. Furthermore, cholera-toxin-catalyzed ADP-ribosylation of both 43-kDa and 47-kDa alpha s was disproportionately increased ninefold and threefold, respectively, possibly reflecting the increased production of an ADP-ribosylation factor in the differentiated cells. The small GTP-binding protein Ha-ras was reduced by 50%, whereas rab1 and other small GTP-binding proteins tentatively identified as rab-isoforms (ras-homologous gene products from brain) were increased by 100% and 70%, respectively. Since the total protein content of 3T3-L1 cells was increased threefold after differentiation, the observed increase of the 43-kDa alpha s, rab1 and of the other rab isoforms was eightfold, sixfold and fivefold, respectively, when normalized/cell count. With the exception of the rab isoforms, all GTP-binding proteins were predominantly, if not exclusively, located in the plasma membrane; comparable amounts of the rab isoforms were found in plasma membranes and low-density microsomes. Insulin induced the characteristic redistribution of glucose transporters GLUT4 from low-density microsomes to the plasma membranes, but failed to alter the subcellular distribution of any of the GTP-binding proteins investigated. These data suggest that the increase in the abundance of the 43-kDa alpha s subunit and of several rab isoforms might be related to specific functions of the adipocyte-like phenotype, but that none of the investigated guanine-nucleotide-binding regulatory (G)-proteins appears to be tightly associated with the GLUT4.

Pyrrolo-pyrimidones of the general structure 1 were synthesized and evaluated for their potential as MK2 inhibitors. Potent derivatives were discovered which inhibit MK2 in the nanomolar range and show potent inhibition of cytokine release from LPS-stimulated monocytes. These derivatives were shown to inhibit phosphorylation of hsp27, a downstream target of MK2 and are modestly selective in a panel of 28 kinases.

UCP3 is a mitochondrial membrane protein expressed in humans selectively in skeletal muscle. To determine the mechanisms by which UCP3 plays a role in regulating glucose metabolism, we expressed human UCP3 in L6 myotubes by adenovirus-mediated gene transfer and in H(9)C(2) cardiomyoblasts by stable transfection with a tetracycline-repressible UCP3 construct. Expression of UCP3 in L6 myotubes increased 2-deoxyglucose uptake 2-fold and cell surface GLUT4 2.3-fold, thereby reaching maximally insulin-stimulated levels in control myotubes. Wortmannin, LY 294002, or the tyrosine kinase inhibitor genistein abolished the effect of UCP3 on glucose uptake, and wortmannin inhibited UCP3-induced GLUT4 cell surface recruitment. UCP3 overexpression increased phosphotyrosine-associated phosphoinositide 3-kinase (PI3K) activity 2.2-fold compared with control cells (p < 0.05). UCP3 overexpression increased lactate release 1.5- to 2-fold above control cells, indicating increased glucose metabolism. In H(9)C(2) cardiomyoblasts stably transfected with UCP3 under control of a tetracycline-repressible promotor, removal of doxycycline resulted in detectable levels of UCP3 at 12 h and 2.2-fold induction at 7 days compared with 12 h. In parallel, glucose transport increased 1.3- and 2-fold at 12 h and 7 days, respectively, and the stimulation was inhibited by wortmannin or genistein. p85 association with membranes was increased 5.5-fold and phosphotyrosine-associated PI3K activity 3.8-fold. In contrast, overexpression of UCP3 in 3T3-L1 adipocytes did not alter glucose uptake, suggesting tissue-specific effects of human UCP3. Thus, UCP3 stimulates glucose transport and GLUT4 translocation to the cell surface in cardiac and skeletal muscle cells by activating a PI3K dependent pathway.

The cDNA of a novel, ubiquitously expressed protein kinase (Dyrk) was cloned from a rat brain cDNA library. The deduced amino acid sequence (763 amino acids) contains a catalytic domain that is only distantly related to that of other mammalian protein kinases. Its closest relative is the protein kinase Mnb of Drosophila, which is presumably involved in postembryonic neurogenesis (85% identical amino acids within the catalytic domain). Outside the catalytic domain, the sequence comprises several striking structural features: a bipartite nuclear translocation signal, a tyrosine-rich hydrophilic motif flanking the nuclear localization signal, a PEST region, a repeat of 13 histidines, a repeat of 17 serine/threonine residues, and an alternatively spliced insertion of nine codons. A recombinant glutathione S-transferase-Dyrk fusion protein catalyzed autophosphorylation and histone phosphorylation on tyrosine and serine/threonine residues with an apparent K of approximately 3.4 μM. Exchange of two tyrosine residues in the “activation loop” between subdomains VII and VIII for phenylalanine almost completely suppressed the activity and tyrosine autophosphorylation of Dyrk. Tyrosine autophosphorylation was also reduced by exchange of the tyrosine (Tyr-219) in a tyrosine phosphorylation consensus motif. The data suggest that Dyrk is a dual specificity protein kinase that is regulated by tyrosine phosphorylation in the activation loop and might be a component of a signaling pathway regulating nuclear functions.

A polymerase chain reaction-based cloning approach was employed in order to identify ADP-ribosylation factors (ARF) in murine 3T3-L1 cells and to study their expression before and after differentiation of cells to the adipocyte-like phenotype. Partial sequences comprising the effector domains of ARF were amplified with degenerate primers and cloned. Five of these sequences were identified as murine homologues of known human ADP-ribosylation factors (ARF 1, 2, 4, 5, and 6). In addition, partial sequences of two previously unknown isoforms were found, and complete cDNA clones were isolated from a rat fat cell library and were sequenced. Both sequences harbor a putative myristoylation site in position 2, the known consensus sequences presumably involved in GTP binding and hydrolysis, and lack cysteine residues in the C terminus. Their amino acid sequences share a 56 and 41% identity, respectively, with human ARF 1. Based on a comparison with the known ARF isoforms, the first clone appears to represent the mammalian homologue of a known sequence from Drosophila (dARL 1, 79% identity) and was therefore designated rARL 1. The second clone resembled none of the known ARF-like proteins and was designated rARL 4. mRNA of ARL 4 was undetectable in the fibroblasts but abundant in the adipocyte-like phenotype, its expression starting on day 6 of the differentiation. In contrast, ARF 1, 2, and 5 were unaltered by differentiation of the 3T3-L1 cells; mRNA levels of ARF 6, and also of ARL 1 and ARF 4, were reduced after differentiation. It is suggested that the function of ARL 4 is related to the adipocyte-like phenotype of 3T3-L1 cells.