Disrupting Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Latent Replication with a Small Molecule Inhibitor.

Kaposi's sarcoma-associated herpesvirus (KSHV) can establish latent lifelong infections in infected individuals. During viral latency, the latency-associated nuclear antigen (LANA) mediates the replication of the latent viral genome in dividing cells and tethers them to mitotic chromosomes, thus ensuring their partitioning into daughter cells during mitosis. This study aims to inhibit Kaposi's sarcoma-associated herpesvirus (KSHV) latent replication by targeting the LANA-DNA interaction using small molecular entities. Drawing from first-generation inhibitors and using growth vectors identified through STD-NMR, we expanded these compounds using Suzuki-Miyaura cross-coupling. This led to a deeper understanding of SAR achieved by microscale thermophoresis (MST) measurements and cell-free tests via electrophoretic mobility shift assays (EMSA). Our most potent compounds successfully inhibit LANA-mediated replication in cell-based assays and demonstrate favorable in vitro ADMET-profiles, including suitable metabolic stability, Caco-2 permeability, and cytotoxicity. These compounds could serve as qualified leads for the future refinement of small molecule inhibitors of KSHV latent replication.

Innate activation of human neutrophils and neutrophil-like cells by the pro-inflammatory bacterial metabolite ADP-heptose and Helicobacter pylori.

Lipopolysaccharide inner core heptose metabolites, including ADP-heptose, play a substantial role in the activation of cell-autonomous innate immune responses in eukaryotic cells, via the ALPK1-TIFA signaling pathway, as demonstrated for various pathogenic bacteria. The important role of LPS heptose metabolites during Helicobacter pylori infection of the human gastric niche has been demonstrated for gastric epithelial cells and macrophages, while the role of heptose metabolites on human neutrophils has not been investigated. In this study, we aimed to gain a better understanding of the activation potential of bacterial heptose metabolites for human neutrophil cells. To do so, we used pure ADP-heptose and, as a bacterial model, H. pylori, which can transport heptose metabolites into the human host cell via the Cag Type 4 Secretion System (CagT4SS). Main questions were how bacterial heptose metabolites impact on the pro-inflammatory activation, alone and in the bacterial context, and how they influence maturation of human neutrophils. Results of the present study demonstrated that neutrophils respond with high sensitivity to pure heptose metabolites, and that global regulation networks and neutrophil maturation are influenced by heptose exposure. Furthermore, activation of human neutrophils by live H. pylori is strongly impacted by the presence of LPS heptose metabolites and the functionality of its CagT4SS. Similar activities were determined in cell culture neutrophils of different maturation states and in human primary neutrophils. In conclusion, we demonstrated that specific heptose metabolites or bacteria producing heptoses exhibit a strong activity on cell-autonomous innate responses of human neutrophils.

Kaposi sarcoma-associated herpesvirus latency-associated nuclear antigen: more than a key mediator of viral persistence.

Kaposi sarcoma-associated herpesvirus (KSHV), or human herpesvirus-8, is an oncogenic herpesvirus. Its latency-associated nuclear antigen (LANA) is essential for the persistence of KSHV in latently infected cells. LANA mediates replication of the latent viral genome during the S phase of a dividing cell and partitions episomes to daughter cells by attaching them to mitotic chromosomes. It also mediates the establishment of latency in newly infected cells through epigenetic mechanisms and suppresses the activation of the productive replication cycle. Furthermore, LANA promotes the proliferation of infected cell by acting as a transcriptional regulator and by modulating the cellular proteome through the recruitment of several cellular ubiquitin ligases. Finally, LANA interferes with the innate and adaptive immune system to facilitate the immune escape of infected cells.

Innate immune activation and modulatory factors of Helicobacter pylori towards phagocytic and nonphagocytic cells.

Helicobacter pylori is an intriguing obligate host-associated human pathogen with a specific host interaction biology, which has been shaped by thousands of years of host-pathogen coevolution. Molecular mechanisms of interaction of H. pylori with the local immune cells in the human system are less well defined than epithelial cell interactions, although various myeloid cells, including neutrophils and other phagocytes, are locally present or attracted to the sites of infection and interact with H. pylori. We have recently addressed the question of novel bacterial innate immune stimuli, including bacterial cell envelope metabolites, that can activate and modulate cell responses via the H. pylori Cag type IV secretion system. This review article gives an overview of what is currently known about the interaction modes and mechanisms of H. pylori with diverse human cell types, with a focus on bacterial metabolites and cells of the myeloid lineage including phagocytic and antigen-presenting cells.

Discovery of ultrapotent broadly neutralizing antibodies from SARS-CoV-2 elite neutralizers.

A fraction of COVID-19 convalescent individuals mount a potent antibody response to SARS-CoV-2 with cross-reactivity to SARS-CoV-1. To uncover their humoral response in detail, we performed single B cell analysis from 10 SARS-CoV-2 elite neutralizers. We isolated and analyzed 126 monoclonal antibodies, many of which were sarbecovirus cross-reactive, with some displaying merbecovirus- and embecovirus-reactivity. Several isolated broadly neutralizing antibodies were effective against B.1.1.7, B.1.351, B.1.429, B.1.617, and B.1.617.2 variants and 19 prominent potential escape sites. Furthermore, assembly of 716,806 SARS-CoV-2 sequences predicted emerging escape variants, which were also effectively neutralized. One of these broadly neutralizing potent antibodies, R40-1G8, is a IGHV3-53 RBD-class-1 antibody. Remarkably, cryo-EM analysis revealed that R40-1G8 has a flexible binding mode, targeting both "up" and "down" conformations of the RBD. Given the threat of emerging SARS-CoV-2 variants, we demonstrate that elite neutralizers are a valuable source for isolating ultrapotent antibody candidates to prevent and treat SARS-CoV-2 infection.

Contribution of Heptose Metabolites and the cag Pathogenicity Island to the Activation of Monocytes/Macrophages by Helicobacter pylori.

The human gastric pathogen Helicobacter pylori activates human epithelial cells by a particular combination of mechanisms, including NOD1 and ALPK1-TIFA activation. These mechanisms are characterized by a strong participation of the bacterial cag pathogenicity island, which forms a type IV secretion system (CagT4SS) that enables the bacteria to transport proteins and diverse bacterial metabolites, including DNA, glycans, and cell wall components, into human host cells. Building on previous findings, we sought to determine the contribution of lipopolysaccharide inner core heptose metabolites (ADP-heptose) in the activation of human phagocytic cells by H. pylori. Using human monocyte/macrophage-like Thp-1 cells and human primary monocytes and macrophages, we were able to determine that a substantial part of early phagocytic cell activation, including NF-κB activation and IL-8 production, by live H. pylori is triggered by bacterial heptose metabolites. This effect was very pronounced in Thp-1 cells exposed to bacterial purified lysates or pure ADP-heptose, in the absence of other bacterial MAMPs, and was significantly reduced upon TIFA knock-down. Pure ADP-heptose on its own was able to strongly activate Thp-1 cells and human primary monocytes/macrophages. Comprehensive transcriptome analysis of Thp-1 cells co-incubated with live H. pylori or pure ADP-heptose confirmed a signature of ADP-heptose-dependent transcript activation in monocyte/macrophages. Bacterial enzyme-treated lysates (ETL) and pure ADP-heptose-dependent activation differentiated monocytes into macrophages of predominantly M1 type. In Thp-1 cells, the active CagT4SS was less required for the heptose-induced proinflammatory response than in epithelial cells, while active heptose biosynthesis or pure ADP-heptose was required and sufficient for their early innate response and NF-κB activation. The present data suggest that early activation and maturation of incoming and resident phagocytic cells (monocytes, macrophages) in the H. pylori-colonized stomach strongly depend on bacterial LPS inner core heptose metabolites, also with a significant contribution of an active CagT4SS.

Hit-to-lead optimization of a latency-associated nuclear antigen inhibitor against Kaposi's sarcoma-associated herpesvirus infections.

The Latency-associated nuclear antigen (LANA) plays a central role for the latent persistence of the Kaposi's Sarcoma Herpesvirus (KSHV) in the human host and helps to establish lifelong infections. Herein, we report our efforts towards hit-to-lead generation starting from a previously discovered LANA-DNA inhibitor. By tethering the viral genome to the host nucleosomes, LANA ensures the segregation and persistence of the viral DNA during mitosis. LANA is also required for the replication of the latent viral episome during the S phase of the cell cycle. We aim to inhibit the interaction between LANA and the viral genome to prevent the latent persistence of KSHV in the host organism. Medicinal chemistry-driven optimization studies and structure-activity-relationship investigation led to the discovery of an improved LANA inhibitor. The functional activity of our compounds was evaluated using a fluorescence polarization (FP)-based interaction inhibition assay and electrophoretic mobility shift assay (EMSA). Even though a crystal structure of the ligand protein complex was not available, we successfully conducted hit optimization toward a low micromolar protein-nucleic acid-interaction inhibitor. Additionally, we applied STD-NMR studies to corroborate target binding and to gain insights into the binding orientation of our most potent inhibitor, providing opportunities for further rational design of more efficient LANA-targeting anti KSHV agents in future studies.

Fragment-Based Discovery of a Qualified Hit Targeting the Latency-Associated Nuclear Antigen of the Oncogenic Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8.

The latency-associated nuclear antigen (LANA) is required for latent replication and persistence of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8. It acts via replicating and tethering the virus episome to the host chromatin and exerts other functions. We conceived a new approach for the discovery of antiviral drugs to inhibit the interaction between LANA and the viral genome. We applied a biophysical screening cascade and identified the first LANA binders from small, structurally diverse compound libraries. Starting from a fragment-sized scaffold, we generated optimized hits via fragment growing using a dedicated fluorescence-polarization-based assay as the structure-activity-relationship driver. We improved compound potency to the double-digit micromolar range. Importantly, we qualified the resulting hit through orthogonal methods employing EMSA, STD-NMR, and MST methodologies. This optimized hit provides an ideal starting point for subsequent hit-to-lead campaigns providing evident target-binding, suitable ligand efficiencies, and favorable physicochemical properties.

Helicobacter pylori modulates host cell responses by CagT4SS-dependent translocation of an intermediate metabolite of LPS inner core heptose biosynthesis.

Highly virulent Helicobacter pylori cause proinflammatory signaling inducing the transcriptional activation and secretion of cytokines such as IL-8 in epithelial cells. Responsible in part for this signaling is the cag pathogenicity island (cagPAI) that codetermines the risk for pathological sequelae of an H. pylori infection such as gastric cancer. The Cag type IV secretion system (CagT4SS), encoded on the cagPAI, can translocate various molecules into cells, the effector protein CagA, peptidoglycan metabolites and DNA. Although these transported molecules are known to contribute to cellular responses to some extent, a major part of the cagPAI-induced signaling leading to IL-8 secretion remains unexplained. We report here that biosynthesis of heptose-1,7-bisphosphate (HBP), an important intermediate metabolite of LPS inner heptose core, contributes in a major way to the H. pylori cagPAI-dependent induction of proinflammatory signaling and IL-8 secretion in human epithelial cells. Mutants defective in the genes required for synthesis of HBP exhibited a more than 95% reduction of IL-8 induction and impaired CagT4SS-dependent cellular signaling. The loss of HBP biosynthesis did not abolish the ability to translocate CagA. The human cellular adaptor TIFA, which was described before to mediate HBP-dependent activity in other Gram-negative bacteria, was crucial in the cagPAI- and HBP pathway-induced responses by H. pylori in different cell types. The active metabolite was present in H. pylori lysates but not enriched in bacterial supernatants. These novel results advance our mechanistic understanding of H. pylori cagPAI-dependent signaling mediated by intracellular pattern recognition receptors. They will also allow to better dissect immunomodulatory activities by H. pylori and to improve the possibilities of intervention in cagPAI- and inflammation-driven cancerogenesis.

Cell-specific regulation of nucleic acid sensor cascades: a controlling interest in the antiviral response.

In this study, we examined the capacities of non-antigen-presenting cell types to propagate antiviral signals following infection with recombinant adenovirus or by direct nucleic acid transfection. Three murine cell lines (RAW264.7 macrophages as a positive control, FL83B hepatocytes, and MS1 endothelial cells) were assessed following exposure to adenovirus, DNA, or RNA ligands. Based on primary (interferon response factor 3 [IRF3] phosphorylation) and secondary (STAT1/2 phosphorylation) response markers, we found each cell line presented a unique response profile: RAW cells were highly responsive, MS1 cells were modified in their response, and FL83B cells were essentially nonresponsive. Comparative reverse transcription-quantitative PCR (RT-qPCR) of nucleic acid sensing components revealed major differences between the three cell types. A prominent difference was at the level of adaptor molecules; TRIF, MyD88, MAVS, and STING. TRIF was absent in MS1 and FL83B cells, whereas MyD88 levels were diminished in FL83B hepatocytes. These differences resulted in compromised TLR-mediated activation. While the cytosolic adaptor MAVS was well represented in all cell lines, the DNA adaptor STING was deficient in FL83B hepatocytes (down by nearly 3 log units). The absence of STING provides an explanation for the lack of DNA responsiveness in these cells. This hypothesis was confirmed by acquisition of IRF3 activation in Flag-STING FL83B cells following DNA transfection. To consolidate the central role of adaptors in MS1 endothelial cells, short hairpin RNA (shRNA) knockdown of STING and MAVS resulted in a ligand-specific loss of IRF3 responsiveness. In contrast to the requirement for specific adaptor proteins, a requirement for a specific DNA sensor (AIM2, DDx41, or p204) in the IRF3 activation response was not detected by shRNA knockdown in MS1 cells. The data reveal that cell-specific regulation of nucleic acid sensing cascade components influences antiviral recognition responses, that controlling levels of adaptor molecules is a recurring strategy in regulating antiviral recognition response functions, and that comparative RT-qPCR has predictive value for antiviral/innate response functions in these cells.

Sensing adenovirus infection: activation of interferon regulatory factor 3 in RAW 264.7 cells.

We have used the RAW 264.7 murine macrophage-like cell line as a platform to characterize the recognition and early signaling response to recombinant adenoviral vectors (rAdV). Infection of RAW 264.7 cells triggers an early response (2 to 6 h postinfection) that includes phosphorylation of the interferon (IFN) response factor 3 (IRF3) transcription factor, upregulation of IRF3 primary response genes (interferon-stimulated gene 56 [ISG56], beta IFN [IFN-ß]), and subsequent type I IFN secondary signaling (STAT1/2 phosphorylation). Using short hairpin RNA (shRNA) lentiviral vectors, we show an essential role for Tank binding kinase 1 (TBK1) in this pathway. Data also support a role for STING (MITA) as an adaptor functioning in response to rAdV infection. Using UV/psoralen (Ps)-inactivated virus to block viral transcription, Ps-inactivated virus stimulated primary (IRF3) and secondary (STAT1/2) activation events to the same degree as untreated virus. IRF3 phosphorylation was not blocked in RAW 264.7 cells pretreated with the RNA polymerase III inhibitor ML60218. However, they were compromised in the type I IFN-dependent secondary response (phosphorylation of STAT1/STAT2). At 24 h postinfection, ML60218-treated cells were compromised in the overall antiviral response. Therefore, initial sensing of rAdV or viral DNA (vDNA) does not depend on viral template transcription, but ML60218 treatment influences cellular cascades required for an antiviral response to rAdV. Using overexpression or knockdown assays, we examined how four DNA sensors influence the antiviral response. Knockdown of DNA Activator of Interferon (DAI) and p204, the murine ortholog to IFI16, had minimal influence on IRF3 phosphorylation. However, knockdown of absent in melanoma 2 (AIM2) and the helicase DDX41 resulted in diminished levels of (pser388)IRF3 following rAdV infection. Based on these data, multiple DNA sensors contribute to an antiviral DNA recognition response, leading to TBK1-dependent IRF3 phosphorylation in RAW 264.7 cells.