Non-canonical Inhibitors Research Articles

Different Expression Levels of DLK2 Inhibit NOTCH Signaling and Inversely Modulate MDA-MB-231 Breast Cancer Tumor Growth In Vivo.

NOTCH signaling is implicated in the development of breast cancer tumors. DLK2, a non-canonical inhibitor of NOTCH signaling, was previously shown to be involved in skin and breast cancer. In this work, we studied whether different levels of DLK2 expression influenced the breast cancer characteristics of MDA-MB-231 cells. We found that DLK2 overexpression inhibited NOTCH activation in a dose-dependent manner. Moreover, depending on the level of inhibition of NOTCH1 activation generated by different levels of DLK2 expression, cell proliferation, cell cycle dynamics, cell apoptosis, cell migration, and tumor growth in vivo were affected in opposite directions. Low levels of DLK2 expression produced a slight inhibition of NOTCH1 activation, and enhanced MDA-MB-231 cell invasion in vitro and cell proliferation both in vitro and in vivo. In contrast, MDA-MB-231 cells expressing elevated levels of DLK2 showed a strong inhibition of NOTCH1 activation, decreased cell proliferation, increased cell apoptosis, and were unable to generate tumors in vivo. In addition, DLK2 expression levels also affected some members of other cell signaling pathways implicated in cancer, such as ERK1/2 MAPK, AKT, and rpS6 kinases. Our data support an important role of DLK2 as a protein that can finely regulate NOTCH signaling and affect the tumor properties and growth dynamics of MDA-MB-231 breast cancer cells.

An Overview of Next-Generation Androgen Receptor-Targeted Therapeutics in Development for the Treatment of Prostate Cancer.

Traditional endocrine therapy for prostate cancer (PCa) has been directed at suppression of the androgen receptor (AR) signaling axis since Huggins et al. discovered that diethylstilbestrol (DES; an estrogen) produced chemical castration and PCa tumor regression. Androgen deprivation therapy (ADT) still remains the first-line PCa therapy. Insufficiency of ADT over time leads to castration-resistant PCa (CRPC) in which the AR axis is still active, despite castrate levels of circulating androgens. Despite the approval and use of multiple generations of competitive AR antagonists (antiandrogens), antiandrogen resistance emerges rapidly in CRPC due to several mechanisms, mostly converging in the AR axis. Recent evidence from multiple groups have defined noncompetitive or noncanonical direct binding sites on AR that can be targeted to inhibit the AR axis. This review discusses new developments in the PCa treatment paradigm that includes the next-generation molecules to noncanonical sites, proteolysis targeting chimera (PROTAC), or noncanonical N-terminal domain (NTD)-binding of selective AR degraders (SARDs). A few lead compounds targeting each of these novel noncanonical sites or with SARD activity are discussed. Many of these ligands are still in preclinical development, and a few early clinical leads have emerged, but successful late-stage clinical data are still lacking. The breadth and diversity of targets provide hope that optimized noncanonical inhibitors and/or SARDs will be able to overcome antiandrogen-resistant CRPC.

Druggable Transient Pockets in Protein Kinases

Drug discovery using small molecule inhibitors is reaching a stalemate due to low selectivity, adverse off-target effects and inevitable failures in clinical trials. Conventional chemical screening methods may miss potent small molecules because of their use of simple but outdated kits composed of recombinant enzyme proteins. Non-canonical inhibitors targeting a hidden pocket in a protein have received considerable research attention. Kii and colleagues identified an inhibitor targeting a transient pocket in the kinase DYRK1A during its folding process and termed it FINDY. FINDY exhibits a unique inhibitory profile; that is, FINDY does not inhibit the fully folded form of DYRK1A, indicating that the FINDY-binding pocket is hidden in the folded form. This intriguing pocket opens during the folding process and then closes upon completion of folding. In this review, we discuss previously established kinase inhibitors and their inhibitory mechanisms in comparison with FINDY. We also compare the inhibitory mechanisms with the growing concept of “cryptic inhibitor-binding sites.” These sites are buried on the inhibitor-unbound surface but become apparent when the inhibitor is bound. In addition, an alternative method based on cell-free protein synthesis of protein kinases may allow the discovery of small molecules that occupy these mysterious binding sites. Transitional folding intermediates would become alternative targets in drug discovery, enabling the efficient development of potent kinase inhibitors.

Androgen Receptor Is a Non-canonical Inhibitor of Wild-Type and Mutant Estrogen Receptors in Hormone Receptor-Positive Breast Cancers.

SummarySustained treatment of estrogen receptor (ER)-positive breast cancer with ER-targeting drugs results in ER mutations and refractory unresponsive cancers. Androgen receptor (AR), which is expressed in 80%–95% of ER-positive breast cancers, could serve as an alternate therapeutic target. Although AR agonists were used in the past to treat breast cancer, their use is currently infrequent due to virilizing side effects. Discovery of tissue-selective AR modulators (SARMs) has renewed interest in using AR agonists to treat breast cancer. Using translational models, we show that AR agonist and SARM, but not antagonist, inhibit the proliferation and growth of ER-positive breast cancer cells, patient-derived tissues, and patient-derived xenografts (PDX). Ligand-activated AR inhibits wild-type and mutant ER activity by reprogramming the ER and FOXA1 cistrome and rendering tumor growth inhibition. These findings suggest that ligand-activated AR may function as a non-canonical inhibitor of ER and that AR agonists may offer a safe and effective treatment for ER-positive breast cancer.

1.45 Å resolution structure of SRPN18 from the malaria vector Anopheles gambiae.

Serine protease inhibitors (serpins) in insects function within development, wound healing and immunity. The genome of the African malaria vector, Anopheles gambiae, encodes 23 distinct serpin proteins, several of which are implicated in disease-relevant physiological responses. A. gambiae serpin 18 (SRPN18) was previously categorized as non-inhibitory based on the sequence of its reactive-center loop (RCL), a region responsible for targeting and initiating protease inhibition. The crystal structure of A. gambiae SRPN18 was determined to a resolution of 1.45 Å, including nearly the entire RCL in one of the two molecules in the asymmetric unit. The structure reveals that the SRPN18 RCL is extremely short and constricted, a feature associated with noncanonical inhibitors or non-inhibitory serpin superfamily members. Furthermore, the SRPN18 RCL does not contain a suitable protease target site and contains a large number of prolines. The SRPN18 structure therefore reveals a unique RCL architecture among the highly conserved serpin fold.

Increased Wnt Signaling and Reduced Viability in a Neuronal Model of Progranulin-Deficient Frontotemporal Lobar Degeneration.

Progranulin (PGRN) deficiency is considered the major cause of frontotemporal lobar degeneration with TDP-43 protein inclusions (FTLD-TDP). Recent work unveiled a relationship between Wnt signaling and PGRN in cellular models of FTLD and cells of patients carrying loss-of-function GRN mutations. This study was undertaken to explore the relationship between PGRN deficit and Wnt signaling in the regulation of survival of GRN knockdown neuroblastoma SH-SY5Y cells (GRN KD). We report here that both canonical and noncanonical Wnt signaling cascades are overactivated in GRN KD cells. We detected increased expression levels of Wnt1 and Wnt5a ligands of the Frizzled receptors, as well as evidence for increased signaling of the Wnt/β-catenin and Wnt/Ca2+ cascades in PGRN deficient cells, such as increased nuclear content of β-catenin and higher levels of cyclin D1, or increased levels of the active form of the NFAT1 transcription factor, respectively. Upregulation of either Wnt/β-catenin or Wnt/Ca2+ signaling in GRN KD cells leads to the stimulation of BrdU incorporation into DNA, hyperphosphorylation of the pRb family of proteins and reduced cell viability over time. Blocking the Wnt cascades by specific canonical or noncanonical inhibitors of Wnt-dependent signaling, normalized the rate of DNA synthesis, and what it is more important restored the viability of GRN KD cells. Our results suggest an important role of Wnt activation inducing cell cycle disturbance-mediated neuronal loss in the pathogenesis of PGRN deficiency-linked FTLD-TDP. Therefore, it is plausible that modulation of Wnt signaling could be a promising strategy for developing of new disease-modifying treatments for FTLD-TDP.

Abstract 15544: GRK2 RGS Domain-Specific Interaction With Gαq Inhibits Hypertrophy and Cardiac Dysfunction During Pressure Overload

G protein-coupled receptor (GPCR) kinases (GRK) play a critical role in cardiac function through regulation of GPCR activity. Canonically, GRK2 phosphorylates active GPCRs leading to desensitization and down-regulation of the receptors to regulate signaling in the heart. Mounting evidence, however, supports non-canonical regulation of cardiac function by GRK2 through a dynamic “interactome” in which GRK2 can uncouple GPCRs via novel protein-protein interactions. Several GRK2 interacting partners are important for adaptive and maladaptive myocyte growth including Gq, the signaling trigger for maladaptive cardiac hypertrophy, leading to HF. Importantly, GRK2 contains a putative amino-terminal Regulator of G protein Signaling (RGS) domain, that directly interacts with Gq and appears to inhibit signaling without altering Gq enzymatic activity. In the present study we generated transgenic (Tg) mice with cardiac-specific expression of the RGS domain of GRK2 (βARKrgs) and subjected them to a trans-aortic constriction (TAC) model of pressure overload hypertrophy. The βARKrgs Tg mice exhibited a marked anti-hypertrophic effect, with reduced left ventricular wall thickness and hypertrophic gene expression relative to their littermate controls. Use of genetically engineered lines demonstrated that this was through an interaction of the βARKrgs peptide with Gq-mediated signaling that inhibited hypertrophy as well as the progression to heart failure (HF). These data support our hypothesis that the RGS domain of GRK2 may serve as a non-canonical inhibitor of Gq-mediated hypertrophic signaling in the heart and highlight how this research may pave the way for novel GRK2-based therapeutic approaches to prevent hypertrophy and HF.

Transforming growth factor type-β inhibits Mas receptor expression in fibroblasts but not in myoblasts or differentiated myotubes; Relevance to fibrosis associated to muscular dystrophies.

Duchenne muscular dystrophy is a genetic disorder characterized by myofiber degeneration, muscle weakness, and increased fibrosis. Transforming growth factor type-β (TGF-β), a central mediator of fibrosis, is upregulated in fibrotic diseases. Angiotensin-(1-7) [Ang-(1-7)] is a peptide with actions that oppose those of angiotensin-II (Ang II). Ang-(1-7) effects are mediated by the Mas receptor. Treatment with Ang-(1-7) produce positive effects in the mdx mouse, normalizing skeletal muscle architecture, decreasing local fibrosis, and fibroblasts, and improving muscle function. Mdx mice deficient for the Mas receptor showed the opposite effects. To identify the cell type(s) responsible for Mas receptor expression, and to characterize whether profibrotic effectors had any effect on its expression, we determined the effect of profibrotic agents on Mas expression. TGF-β, but not connective tissue growth factor or Ang-II, reduced the expression of Mas receptor in fibroblasts isolated from skeletal muscle cells and fibroblasts from two established cell lines. In contrast, no effects were observed in myoblasts and differentiated myotubes. This inhibition was mediated by the Smad-dependent (canonical) and the PI3K and MEK1/2 (noncanonical) TGF-β signaling pathways. When both canonical and noncanonical inhibitors of the TGF-β-dependent pathways were added together, the inhibitory effect of TGF-β on Mas expression was lost. The decrease in Mas receptor induced by TGF-β in fibroblasts reduced the Ang-(1-7) mediated stimulation of phosphorylation of AKT pathway proteins. These results suggest that reduction of Mas receptor in fibroblasts, by TGF-β, could increase the fibrotic phenotype observed in dystrophic skeletal muscle decreasing the beneficial effect of Ang-(1-7).

Abstract 70: Domain-Specific Roles for GRK2 in Cardiac Hypertrophy and Heart Failure

During heart failure (HF), cardiac levels and activity of the G protein-coupled receptor (GPCR) kinase (GRK) GRK2 are elevated, increasing phosphorylation, desensitization and down-regulation of β-adrenergic receptors (βARs) and other cardiac GPCRs. Increased GRK2 participates in adverse remodeling and contractile dysfunction during HF, while inhibition via a carboxy-terminal peptide, βARKct, enhances heart function and can prevent HF development. Mounting evidence supports the idea of a dynamic “interactome” in which GRK2 can uncouple GPCRs via novel protein-protein interactions. Several novel GRK2 interacting partners are important for adaptive and maladaptive myocyte growth including Gq, the signaling trigger for maladaptive cardiac hypertrophy, leading to HF. Importantly, GRK2 contains a putative amino-terminal Regulator of G protein Signaling (RGS) domain (βARK-RGS). This domain directly interacts with Gq and appears to inhibit signaling without altering Gq enzymatic activity. Therefore, this domain may alter hypertrophic responses in the heart and represent a novel role for GRK2 and a potential therapeutic target to limit maladaptive cardiac hypertrophy. We have begun to address this by generation of novel transgenic (Tg) mice with cardiac-specific expression of the RGS domain of GRK2. Using a trans-aortic constriction (TAC) model of pressure overload hypertrophy, we found that expression of βARK-RGS demonstrates anti-hypertrophic effects. Echocardiographic analysis post-TAC revealed reduced left ventricular posterior wall thickness (LVPW) in βARK-RGS compared to non-transgenic littermate controls (NLC) (0.85 vs 1.0 mm LVPWd at 4 weeks). RT-PCR analysis found decreased hypertrophic factor transcripts, such as ANF for which the nearly 18-fold increase post TAC was completely inhibited in βARK-RGS mice. Further, the progression to HF was inhibited in βARK-RGS mice, but not NLCs, 14 weeks post-TAC. While mechanistic characterization is underway, these data support our hypothesis that the RGS domain of GRK2 may serve as a non-canonical inhibitor of Gq-mediated hypertrophic signaling in the heart and highlight how this research may pave the way for novel GRK2-based therapeutic approaches to prevent hypertrophy and HF.

Abstract 075: Novel Role for GRK2 in Cardiac Hypertrophy and Heart Failure

During heart failure (HF), cardiac levels and activity of the G protein-coupled receptor (GPCR) kinase (GRK) GRK2 are elevated, increasing phosphorylation, desensitization and down-regulation of β-adrenergic receptors (βARs) and other cardiac GPCRs. Increased GRK2 has been shown to participate in adverse remodeling and contractile dysfunction during HF, while GRK2 inhibition via a carboxy-terminal peptide, βARKct, enhances heart function and can prevent HF development. Mounting evidence supports the idea of a dynamic GRK2 “interactome” in which GRK2 can uncouple GPCRs via novel protein-protein interactions. Several novel GRK2 interacting partners are important for adaptive and maladaptive myocyte growth including Gq, the signaling trigger for maladaptive cardiac hypertrophy, leading to HF. Importantly, GRK2 contains a putative amino-terminal Regulator of G protein Signaling (RGS) domain (termed βARK-RGS). This domain directly interacts with Gq and appears to inhibit signaling without altering Gq enzymatic activity. Therefore, this domain of GRK2 may alter hypertrophic responses in the heart and represent a novel role for GRK2 and also a potential therapeutic target to limit maladaptive cardiac hypertrophy. We have begun to address this by generation of novel transgenic mice with cardiac-specific expression of the RGS domain of GRK2. Data from mice with cardiac expression of βARK-RGS demonstrate anti-hypertrophic effects in a trans-aortic constriction (TAC) model of pressure overload hypertrophy. Echocardiographic analysis post-TAC revealed reduced left ventricular posterior wall thickness in βARK-RGS compared to non-transgenic littermate controls (1.34 vs 1.57 mm LVPWd at 4 weeks). RT-PCR analysis found decreased hypertrophic factor transcripts, such as ANF for which the nearly 18-fold increase post TAC was completely inhibited in βARK-RGS mice. Other hypertrophic phenotypic markers have been studied and mechanistic characterization is underway. These data support our hypothesis that the RGS domain of GRK2 may serve as a non-canonical inhibitor of Gq-mediated hypertrophic signaling in the heart and highlight how this research may pave the way for novel GRK2-based therapeutic approaches to prevent hypertrophy and HF.

Natural Proteinaceous Inhibitors of Serine Proteases

The deregulated proteolysis is associated with various diseases in humans. Proteases are commonly regarded as the therapeutic targets. Almost one-third of all proteolytic enzymes in humans are serine proteases. This work provides a brief characteristic of the proteinaceous natural inhibitors, mostly of serine proteases. The examples of some classical and recently identified canonical and noncanonical inhibitors as well as serpins are described. Their actual and potential therapeutic applications are discussed. Keywords: Serine proteases, canonical, non-canonical inhibitors, serpins.

Natural Proteinaceous Inhibitors of Serine Proteases

The deregulated proteolysis is associated with various diseases in humans. Proteases are commonly regarded as the therapeutic targets. Almost one-third of all proteolytic enzymes in humans are serine proteases. This work provides a brief characteristic of the proteinaceous natural inhibitors, mostly of serine proteases. The examples of some classical and recently identified canonical and noncanonical inhibitors as well as serpins are described. Their actual and potential therapeutic applications are discussed.

Natural Proteinaceous Inhibitors of Serine Proteases

The deregulated proteolysis is associated with various diseases in humans. Proteases are commonly regarded as the therapeutic targets. Almost one-third of all proteolytic enzymes in humans are serine proteases. This work provides a brief characteristic of the proteinaceous natural inhibitors, mostly of serine proteases. The examples of some classical and recently identified canonical and noncanonical inhibitors as well as serpins are described. Their actual and potential therapeutic applications are discussed.

A TR3/Nur77 Peptide-Based High-Throughput Fluorescence Polarization Screen for Small Molecule Bcl-B Inhibitors

Nuclear receptor TR3/Nur77/NR4A1 binds several antiapoptotic Bcl-2-family proteins (Bcl-B, Bcl-2, Bfl-1) in a non-BH3-dependent manner. A 9-amino-acid peptide derived from full-length TR3 with polyarginine tail (TR3-r8) recapitulates TR3's binding specificity, displaying high affinity for Bcl-B. TR3-r8 peptide was used to screen for small molecule Bcl-B inhibitors. A fluorescence polarization assay (FPA) employing fluorescein isothiocyanate (FITC)-labeled TR3-r8 peptide (FITC-TR3-r8) and Bcl-B protein was optimized, with nonfluorescent TR3-r8 serving to demonstrate reversible, competitive binding. Approximately 50,000 compounds were screened at 3.75 mg/L, yielding 145 reproducible hits with > or =50% FITC-TR3-r8 displacement (a confirmed hit rate of 0.29%). After dose-response analyses and counterscreening with an unrelated FITC-based FPA, 6 candidate compounds remained. Nuclear magnetic resonance (NMR) showed that 2 of these compounds bound Bcl-B, but not glutathione S-transferase (GST) control protein. One Bcl-B-binding compound was unable to displace FITClabeled BH3 peptides from Bcl-B, confirming a unique binding mechanism compared with traditional antagonists of antiapoptotic Bcl-2-family proteins. This compound bound Bcl-B with Kd 1.94 +/- 0.38 microM, as determined by isothermal titration calorimetry. Experiments using Bcl-B overexpressing HeLa cells demonstrated that this compound induced Bcl-B-dependent cell death. The current FPA represents a screen that can identify noncanonical inhibitors of Bcl-2-family proteins.

Delineation of Canonical and Non-Canonical NF-κB Pathways in Multiple Myeloma: Therapeutic Implications.

NF-κB pathway plays a crucial role in the pathogenesis in cancer cells including multiple myeloma (MM). The NF-κB complex is dimer in different combinations of Rel family proteins, including p65 (RelA), RelB, c-Rel, p50 (NF-κB1), and p52 (NF-κB2). Recent studies have revealed that NF-κB activity is mediated via two distinct pathways. In the canonical pathway, NF-kB is typically a heterodimer composed of p50 and p65 subunits. In the non-canonical pathway, NF-kB is typically a heterodimer composed of RelB and p100 subunits. We have shown anti-MM activities of IKKβ inhibitors (PS-1145, MLN120B); however, effects of these agents were modest. Our studies therefore suggest that baseline NF-kB activity in MM cells is not totally dependent on the canonical pathway, and that inhibition of only canonical NF-κB pathway may not be sufficient to block total NF-kB activity. In this study, we therefore hypothesized whether non-canonical inhibitors significantly enhanced NF-κB inhibition induced by canonical inhibitors in MM cells. We first examined baseline NF-κB activity using electrophoretic mobility shift assay (EMSA). NF-κB activity varied between cell lines; for example MM.1S, MM.1R and H929 cells have higher level of NF-κB activity than in RPMI8226, INA6 and OPM2 cells. To define the role of canonical and non-canonical pathway, we next examined protein expression of p50, p65 and p52 NF-κB in these cell lines: p65 was highly expressed in all MM cell lines; however, expression of p50 and 52 is variable. Surprisingly, no detectable or weak expression of p50 was observed in U266, RPMI8226, LR5, H929 and OPM2 cell lines, suggesting that baseline NF-kB activity in these cell lines is not maintained only by the canonical pathway. We then attempted to block non-canonical NF-κB pathway in MM cell lines. Specifically since IKKα and IKKβ are client proteins of heat shock protein (Hsp) 90, we examined whether 17AAG could inhibit expression and/or function of IKKα and IKKβ in MM cells. Importantly, both IKKα and IKKβ were significantly downregulated by 17AAG in MM cell lines. To determine whether downregulation of these IKK proteins by 17AAG was due to inhibition of transcription, we next performed real-time quantitative PCR and no significant inhibition of relative expression of IKKβ was observed by 17AAG treatment, suggesting that downregulation of these proteins was a post transcription event. We further examined whether 17AAG enhanced the effect of IKKβ inhibitor MLN120B on NF-κB activity. Although the inhibitory effect by either MLN120B or 17AAG alone on phosphorylation (p) of IκBα triggered by TNFα was marginal, combination treatment of MLN120B with 17AAG almost completely blocked IκBα, suggesting that this combination synergistically inhibit canonical NF-κB activity in MM cells. Importantly, the combination of MLN120B with 17AAG also significantly blocked baseline and TNFα-triggered NF-κB activity, assessed by EMSA, in MM cells. Finally, 17AAG augmented the growth inhibitory effect of MLN120B in the context of bone marrow stromal cells. Taken together, these results showed that baseline and TNFα-triggered NF-κB activities were completely blocked by this combination treatment, and provide the rationale for its clinical evaluation to induce maximum inhibition of NF-κB activity and improve patient outcome in MM.

Canonical protein inhibitors of serine proteases.

Serine proteases and their natural protein inhibitors are among the most intensively studied protein complexes. About 20 structurally diverse inhibitor families have been identified, comprising alpha-helical, beta sheet, and alpha/beta proteins, and different folds of small disulfide-rich proteins. Three different types of inhibitors can be distinguished based on their mechanism of action: canonical (standard mechanism) and non-canonical inhibitors, and serpins. The canonical inhibitors bind to the enzyme through an exposed convex binding loop, which is complementary to the active site of the enzyme. The mechanism of inhibition in this group is always very similar and resembles that of an ideal substrate. The non-canonical inhibitors interact through their N-terminal segment. There are also extensive secondary interactions outside the active site, contributing significantly to the strength, speed, and specificity of recognition. Serpins, similarly to the canonical inhibitors, interact with their target proteases in a substrate-like manner; however, cleavage of a single peptide bond in the binding loop leads to dramatic structural changes.

Activating Interferons with Noncanonical IKKs

Toll-like receptors (TLRs) initiate immune responses to bacterial and viral pathogens by recognition of conserved patterns presented by the pathogens. The TLR signaling pathways culminate in activation of inflammatory cytokine genes, including stimulation of the expression of the type 1 interferons (IFNα/β). Although all TLRs can signal through the adaptor MyD88, TLR3 and TLR4 continue to activate gene expression in the absence of MyD88 (see Barton and Medzhitov). Fitzgerald et al. investigated, using overexpression studies and interfering RNA (RNAi) techniques, the roles of the noncanonical inhibitor of κB kinase (IKK) members, IKKε and TANK-binding kinase 1 [(TBK1), also known as NF-κB-activating kinase (NAK) and tumor necrosis factor receptor-associated factor 2-associated kinase (T2K)] in the activation of interferon regulatory factor 3 (IRF3)-mediated gene expression. Overexpression of TBK1 or IKKε stimulated IRF3 translocation to the nucleus (traced by the movement of an IRF3-green fluorescent protein fusion construct) and stimulated IRF3-dependent expression of an IFNβ reporter and a reporter containing the interferon-stimulated regulatory element (IRSE) from the gene IFIT2 . In addition, overexpression of TBK1or IKKε stimulated secretion of the cytokine RANTES. Treatment of HEK293 cells or 293 T cells with RNAi against TBK1 or IKKε inhibited stimulation of expression of IFIT2 and the IRF3-dependent IFNβ and IRSE reporters and blocked secretion of RANTES in response to virus infection or double-stranded RNA. Coprecipitation experiments demonstrated an interaction between TBK1, IKKε, and IRF3 or the adaptor TRIF. Furthermore, increased gene expression in response to overexpression of TRIF was inhibited by kinase inactive mutants of TBK1 or IKKε. Thus, the authors suggest that the pathway from TLR to IRF3 proceeds through the TRIF adaptor and involves the noncanonical IKKs, TBK1 and IKKε. G. M. Barton, R. Medzhitov, Linking Toll-like receptors to IFNα/β expression. Nat. Immunol. 4 , 432-433 (2003). [Online Journal] K.A. Fitzgerald, S. M. McWhirter, K. L. Faia, D. C. Rowe, E. Latz, D. T. Golenbock, A. J. Coyle, S.-M. Liao, T. Maniatis, IKKε and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4 , 491-496 (2003). [Online Journal]

Activating Interferons with Noncanonical IKKs

Toll-like receptors (TLRs) initiate immune responses to bacterial and viral pathogens by recognition of conserved patterns presented by the pathogens. The TLR signaling pathways culminate in activation of inflammatory cytokine genes, including stimulation of the expression of the type 1 interferons (IFNα/β). Although all TLRs can signal through the adaptor MyD88, TLR3 and TLR4 continue to activate gene expression in the absence of MyD88 (see Barton and Medzhitov). Fitzgerald et al. investigated, using overexpression studies and interfering RNA (RNAi) techniques, the roles of the noncanonical inhibitor of κB kinase (IKK) members, IKKε and TANK-binding kinase 1 [(TBK1), also known as NF-κB-activating kinase (NAK) and tumor necrosis factor receptor-associated factor 2-associated kinase (T2K)] in the activation of interferon regulatory factor 3 (IRF3)-mediated gene expression. Overexpression of TBK1 or IKKε stimulated IRF3 translocation to the nucleus (traced by the movement of an IRF3-green fluorescent protein fusion construct) and stimulated IRF3-dependent expression of an IFNβ reporter and a reporter containing the interferon-stimulated regulatory element (IRSE) from the gene IFIT2. In addition, overexpression of TBK1or IKKε stimulated secretion of the cytokine RANTES. Treatment of HEK293 cells or 293 T cells with RNAi against TBK1 or IKKε inhibited stimulation of expression of IFIT2 and the IRF3-dependent IFNβ and IRSE reporters and blocked secretion of RANTES in response to virus infection or double-stranded RNA. Coprecipitation experiments demonstrated an interaction between TBK1, IKKε, and IRF3 or the adaptor TRIF. Furthermore, increased gene expression in response to overexpression of TRIF was inhibited by kinase inactive mutants of TBK1 or IKKε. Thus, the authors suggest that the pathway from TLR to IRF3 proceeds through the TRIF adaptor and involves the noncanonical IKKs, TBK1 and IKKε.G. M. Barton, R. Medzhitov, Linking Toll-like receptors to IFNα/β expression. Nat. Immunol. 4, 432-433 (2003). [Online Journal]K.A. Fitzgerald, S. M. McWhirter, K. L. Faia, D. C. Rowe, E. Latz, D. T. Golenbock, A. J. Coyle, S.-M. Liao, T. Maniatis, IKKε and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4, 491-496 (2003). [Online Journal]

Protein inhibitors of serine proteinases.

Serine proteinases and their natural protein inhibitors belong to the most intensively studied models of protein-protein recognition. Protein inhibitors do not form a single group but can be divided into about 20 different families. Global structures of proteins representing different inhibitor families are completely different and comprise alpha-helical proteins, beta-sheet proteins, alpha/beta-proteins and different folds of small disulfide-rich proteins. Three different types of inhibitors can be distinguished: canonical (standard mechanism) inhibitors, non-canonical inhibitors, and serpins. The canonical inhibitor binds to the enzyme through the exposed and convex binding loop, which is complementary to the active site of the enzyme. The mechanism of inhibition in this group is consistently very similar and resembles that of an ideal substrate. Non-canonical inhibitors, originating from blood sucking organisms, specifically block enzymes of the blood clotting cascade. The interaction is mediated through inhibitor N-terminus which binds to the proteinase forming a parallel beta-sheet. There are also extensive secondary interactions which provide an additional buried area and contribute significantly to the strength and specificity of recognition. Serpins are major proteinase inhibitors occurring in plasma. Similarly to canonical inhibitors, serpins interact with their target proteinases in a substrate-like manner. However, in the case of serpins, cleavage of a single peptide bond in a flexible and exposed binding loop leads to dramatic structural changes.