Phillip C. Delekta

Thrombin-dependent NF-κB Activation and Monocyte/Endothelial Adhesion Are Mediated by the CARMA3·Bcl10·MALT1 Signalosome

Thrombin is a potent modulator of endothelial function and, through stimulation of NF-κB, induces endothelial expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). These cell surface adhesion molecules recruit inflammatory cells to the vessel wall and thereby participate in the development of atherosclerosis, which is increasingly recognized as an inflammatory condition. The principal receptor for thrombin on endothelial cells is protease-activated receptor-1 (PAR-1), a member of the G protein-coupled receptor superfamily. Although it is known that PAR-1 signaling to NF-κB depends on initial PKC activation, the subsequent steps leading to stimulation of the canonical NF-κB machinery have remained unclear. Here, we demonstrate that a complex of proteins containing CARMA3, Bcl10, and MALT1 links PAR-1 activation to stimulation of the IκB kinase complex. IκB kinase in turn phosphorylates IκB, leading to its degradation and the release of active NF-κB. Further, we find that although this CARMA3·Bcl10·MALT1 signalosome shares features with a CARMA1-containing signalosome found in lymphocytes, there are significant differences in how the signalosomes communicate with their cognate receptors. Specifically, whereas the CARMA1-containing lymphocyte complex relies on 3-phosphoinositide-dependent protein kinase 1 for assembly and activation, the CARMA3-containing endothelial signalosome functions completely independent of 3-phosphoinositide-dependent protein kinase 1 and instead relies on β-arrestin 2 for assembly. Finally, we show that thrombin-dependent adhesion of monocytes to endothelial cells requires an intact endothelial CARMA3·Bcl10·MALT1 signalosome, underscoring the importance of the signalosome in mediating one of the most significant pro-atherogenic effects of thrombin.

The CARMA3-Bcl10-MALT1 Signalosome Promotes Angiotensin II-dependent Vascular Inflammation and Atherogenesis

The CARMA1, Bcl10, and MALT1 proteins together constitute a signaling complex (CBM signalosome) that mediates antigen-dependent activation of NF-κB in lymphocytes, thereby representing a cornerstone of the adaptive immune response. Although CARMA1 is restricted to cells of the immune system, the analogous CARMA3 protein has a much wider expression pattern. Emerging evidence suggests that CARMA3 can substitute for CARMA1 in non-immune cells to assemble a CARMA3-Bcl10-MALT1 signalosome and mediate G protein-coupled receptor activation of NF-κB. Here we show that one G protein-coupled receptor, the type 1 receptor for angiotensin II, utilizes this mechanism for activation of NF-κB in endothelial and vascular smooth muscle cells, thereby inducing pro-inflammatory signals within the vasculature, a key factor in atherogenesis. Further, we demonstrate that Bcl10-deficient mice are protected from developing angiotensin-dependent atherosclerosis and aortic aneurysms. By uncovering a novel vascular role for the CBM signalosome, these findings illustrate that CBM-dependent signaling has functions outside the realm of adaptive immunity and impacts pathobiology more broadly than previously known.

Discovery of potent broad spectrum antivirals derived from marine actinobacteria

Natural products provide a vast array of chemical structures to explore in the discovery of new medicines. Although secondary metabolites produced by microbes have been developed to treat a variety of diseases, including bacterial and fungal infections, to date there has been limited investigation of natural products with antiviral activity. In this report, we used a phenotypic cell-based replicon assay coupled with an iterative biochemical fractionation process to identify, purify, and characterize antiviral compounds produced by marine microbes. We isolated a compound from Streptomyces kaviengensis, a novel actinomycetes isolated from marine sediments obtained off the coast of New Ireland, Papua New Guinea, which we identified as antimycin A1a. This compound displays potent activity against western equine encephalitis virus in cultured cells with half-maximal inhibitory concentrations of less than 4 nM and a selectivity index of greater than 550. Our efforts also revealed that several antimycin A analogues display antiviral activity, and mechanism of action studies confirmed that these Streptomyces-derived secondary metabolites function by inhibiting the cellular mitochondrial electron transport chain, thereby suppressing de novo pyrimidine synthesis. Furthermore, we found that antimycin A functions as a broad spectrum agent with activity against a wide range of RNA viruses in cultured cells, including members of the Togaviridae, Flaviviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Finally, we demonstrate that antimycin A reduces central nervous system viral titers, improves clinical disease severity, and enhances survival in mice given a lethal challenge with western equine encephalitis virus. Our results provide conclusive validation for using natural product resources derived from marine microbes as source material for antiviral drug discovery, and they indicate that host mitochondrial electron transport is a viable target for the continued development of broadly active antiviral compounds.

MALT1 Protease Activation Triggers Acute Disruption of Endothelial Barrier Integrity via CYLD Cleavage

Microvascular endothelial cells maintain a tight barrier to prevent passage of plasma and circulating immune cells into the extravascular tissue compartment, yet endothelial cells respond rapidly to vasoactive substances, including thrombin, allowing transient paracellular permeability. This response is a cornerstone of acute inflammation, but the mechanisms responsible are still incompletely understood. Here, we demonstrate that thrombin triggers MALT1 to proteolytically cleave cylindromatosis (CYLD). Fragmentation of CYLD results in microtubule disruption and a cascade of events leading to endothelial cell retraction and an acute permeability response. This finding reveals an unexpected role for the MALT1 protease, which previously has been viewed mostly as a driver of pro-inflammatory NF-κB signaling in lymphocytes. Thus, MALT1 not only promotes immune cell activation but also acutely regulates endothelial cell biology, actions that together facilitate tissue inflammation. Pharmacologic inhibition of MALT1 may therefore have synergistic impact by targeting multiple disparate steps in the overall inflammatory response.

Novel Indole-2-Carboxamide Compounds Are Potent Broad-Spectrum Antivirals Active against Western Equine Encephalitis Virus In Vivo

ABSTRACT Neurotropic alphaviruses, including western, eastern, and Venezuelan equine encephalitis viruses, cause serious and potentially fatal central nervous system infections in humans for which no currently approved therapies exist. We previously identified a series of thieno[3,2-b]pyrrole derivatives as novel inhibitors of neurotropic alphavirus replication, using a cell-based phenotypic assay (W. Peng et al., J. Infect. Dis. 199:950–957, 2009, doi:http://dx.doi.org/10.1086/597275), and subsequently developed second- and third-generation indole-2-carboxamide derivatives with improved potency, solubility, and metabolic stability (J. A. Sindac et al., J. Med. Chem. 55:3535–3545, 2012, doi:http://dx.doi.org/10.1021/jm300214e; J. A. Sindac et al., J. Med. Chem. 56:9222–9241, 2013, http://dx.doi.org/10.1021/jm401330r). In this report, we describe the antiviral activity of the most promising third-generation lead compound, CCG205432, and closely related analogs CCG206381 and CCG209023. These compounds have half-maximal inhibitory concentrations of ∼1 μM and selectivity indices of >100 in cell-based assays using western equine encephalitis virus replicons. Furthermore, CCG205432 retains similar potency against fully infectious virus in cultured human neuronal cells. These compounds show broad inhibitory activity against a range of RNA viruses in culture, including members of the Togaviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Although their exact molecular target remains unknown, mechanism-of-action studies reveal that these novel indole-based compounds target a host factor that modulates cap-dependent translation. Finally, we demonstrate that both CCG205432 and CCG209023 dampen clinical disease severity and enhance survival of mice given a lethal western equine encephalitis virus challenge. These studies demonstrate that indole-2-carboxamide compounds are viable candidates for continued preclinical development as inhibitors of neurotropic alphaviruses and, potentially, of other RNA viruses. IMPORTANCE There are currently no approved drugs to treat infections with alphaviruses. We previously identified a novel series of compounds with activity against these potentially devastating pathogens (J. A. Sindac et al., J. Med. Chem. 55:3535–3545, 2012, doi:http://dx.doi.org/10.1021/jm300214e; W. Peng et al., J. Infect. Dis. 199:950–957, 2009, doi:http://dx.doi.org/10.1086/597275; J. A. Sindac et al., J. Med. Chem. 56:9222–9241, 2013, http://dx.doi.org/10.1021/jm401330r). We have now produced third-generation compounds with enhanced potency, and this manuscript provides detailed information on the antiviral activity of these advanced-generation compounds, including activity in an animal model. The results of this study represent a notable achievement in the continued development of this novel class of antiviral inhibitors.

The CARMA3–Bcl10–MALT1 Signalosome Drives NFκB Activation and Promotes Aggressiveness in Angiotensin II Receptor–Positive Breast Cancer

The angiotensin II receptor AGTR1, which mediates vasoconstrictive and inflammatory signaling in vascular disease, is overexpressed aberrantly in some breast cancers. In this study, we established the significance of an AGTR1-responsive NFκB signaling pathway in this breast cancer subset. We documented that AGTR1 overexpression occurred in the luminal A and B subtypes of breast cancer, was mutually exclusive of HER2 expression, and correlated with aggressive features that include increased lymph node metastasis, reduced responsiveness to neoadjuvant therapy, and reduced overall survival. Mechanistically, AGTR1 overexpression directed both ligand-independent and ligand-dependent activation of NFκB, mediated by a signaling pathway that requires the triad of CARMA3, Bcl10, and MALT1 (CBM signalosome). Activation of this pathway drove cancer cell-intrinsic responses that include proliferation, migration, and invasion. In addition, CBM-dependent activation of NFκB elicited cancer cell-extrinsic effects, impacting endothelial cells of the tumor microenvironment to promote tumor angiogenesis. CBM/NFκB signaling in AGTR1+ breast cancer therefore conspires to promote aggressive behavior through pleiotropic effects. Overall, our results point to the prognostic and therapeutic value of identifying AGTR1 overexpression in a subset of HER2-negative breast cancers, and they provide a mechanistic rationale to explore the repurposing of drugs that target angiotensin II-dependent NFκB signaling pathways to improve the treatment of this breast cancer subset.Significance: These findings offer a mechanistic rationale to explore the repurposing of drugs that target angiotensin action to improve the treatment of AGTR1-expressing breast cancers. Cancer Res; 78(5); 1225-40. ©2017 AACR.

Manipulation of host factors optimizes the pathogenesis of western equine encephalitis virus infections in mice for antiviral drug development.

While alphaviruses spread naturally via mosquito vectors, some can also be transmitted as aerosols making them potential bioterrorism agents. One such pathogen, western equine encephalitis virus (WEEV), causes fatal human encephalitis via multiple routes of infection and thus presumably via multiple mechanisms. Although WEEV also produces acute encephalitis in non-human primates, a small animal model that recapitulates features of human disease would be useful for both pathogenesis studies and to evaluate candidate antiviral therapies. We have optimized conditions to infect mice with a low passage isolate of WEEV, thereby allowing detailed investigation of virus tropism, replication, neuroinvasion, and neurovirulence. We find that host factors strongly influence disease outcome, and in particular, that age, gender, and genetic background all have significant effects on disease susceptibility independent of virus tropism or replication within the central nervous system. Our data show that experimental variables can be adjusted in mice to recapitulate disease features known to occur in both non-human primates and humans, thus aiding further study of WEEV pathogenesis and providing a realistic therapeutic window for antiviral drug delivery.

The Combined Use of Alphavirus Replicons and Pseudoinfectious Particles for the Discovery of Antivirals Derived from Natural Products

Alphaviruses are a prominent class of reemergent pathogens due to their globally expanding ranges, potential for lethality, and possible use as bioweapons. The absence of effective treatments for alphaviruses highlights the need for innovative strategies to identify antiviral agents. Primary screens that use noninfectious self-replicating RNAs, termed replicons, have been used to identify potential antiviral compounds for alphaviruses. Only inhibitors of viral genome replication, however, will be identified using replicons, which excludes many other druggable steps in the viral life cycle. To address this limitation, we developed a western equine encephalitis virus pseudoinfectious particle system that reproduces several crucial viral life cycle steps in addition to genome replication. We used this system to screen a library containing ~26,000 extracts derived from marine microbes, and we identified multiple bacterial strains that produce compounds with potential antiviral activity. We subsequently used pseudoinfectious particle and replicon assays in parallel to counterscreen candidate extracts, and followed antiviral activity during biochemical fractionation and purification to differentiate between inhibitors of viral entry and genome replication. This novel process led to the isolation of a known alphavirus entry inhibitor, bafilomycin, thereby validating the approach for the screening and identification of potential antiviral compounds.