Inhibition of p53-MDM2 binding reduces senescent cell abundance and improves the adaptive responses of skeletal muscle from aged mice.

Skeletal muscle adaptation to external stimuli, such as regeneration following injury and hypertrophy in response to resistance exercise, are blunted with advanced age. The accumulation of senescent cells, along with defects in myogenic progenitor cell (MPC) proliferation, have been strongly linked as contributing factors to age-associated impairment in muscle adaptation. p53 plays an integral role in all these processes, as upregulation of p53 causes apoptosis in senescent cells and prevents mitotic catastrophe in MPCs from old mice. The goal of this study was to determine if a novel pharmaceutical agent (BI01), which functions by upregulating p53 through inhibition of binding to MDM2, the primary p53 regulatory protein, improves muscle regeneration and hypertrophy in old mice. BI01 effectively reduced the number of senescent cells in vitro but had no effect on MPC survival or proliferation at a comparable dose. Following repeated oral gavage with 2 mg/kg of BI01 (OS) or vehicle (OV), old mice (24 months) underwent unilateral BaCl2 injury in the tibialis anterior (TA) muscle, with PBS injections serving as controls. After 7 days, satellite cell number was higher in the TA of OS compared to OV mice, as was the expression of genes involved in ATP production. By 35 days, old mice treated with BI01 displayed reduced senescent cell burden, enhanced regeneration (higher muscle mass and fiber cross-sectional area) and restoration of muscle function relative to OV mice. To examine the impact of 2 mg/kg BI01 on muscle hypertrophy, the plantaris muscle was subjected to 28 days of mechanical overload (MOV) in OS and OV mice. In response to MOV, OS mice had larger plantaris muscles and muscle fibers than OV mice, particularly type 2b + x fibers, associated with reduced senescent cells. Together our data show that BI01 is an effective senolytic agent that may also augment muscle metabolism to enhance muscle regeneration and hypertrophy in old mice.

A novel mitochondrial complex I ROS inhibitor partially improves muscle regeneration in adult but not old mice.

It is unclear whether mitochondrial dysfunction and redox stress contribute to impaired age-related muscle regenerative capacity. Here we characterized a novel compound, BI4500, that inhibits the release of reactive oxygen species (ROS) from the quinone site in mitochondrial complex I (site IQ). We tested the hypothesis that ROS release from site IQ contributes to impaired regenerative capacity in aging muscle. Electron transfer system site-specific ROS production was measured in adult and aged mouse isolated muscle mitochondria and permeabilized gastrocnemius fibers. BI4500 inhibited ROS production from site IQ in a concentration-dependent manner (IC50 = âˆ¼985 nM) by inhibiting ROS release without impairing complex I-linked respiration. In vivo BI4500 treatment decreased ROS production from site IQ. Muscle injury and sham injury were induced using barium chloride or vehicle injection to the tibialis anterior (TA) muscle in adult and aged male mice. On the same day as injury, mice began a daily gavage of 30 mg/kg BI4500 (BI) or placebo (PLA). Muscle regeneration (H&E, Sirius Red, Pax7) was measured at 5 and 35 days after injury. Muscle injury increased centrally nucleated fibers (CNFs) and fibrosis with no treatment or age effect. There was a significant age by treatment interaction for CNFs at 5- and 35-days post injury with significantly more CNFs in BI adults compared to PLA adults. Muscle fiber cross-sectional area (CSA) recovered significantly more in adult BI mice (-89 ± 365 µm2) compared to old PLA (-599 ± 153 µm2) and old BI (-535 ± 222 µm2, mean ± SD). In situ TA force recovery was measured 35 days after injury and was not significantly different by age or treatment. Inhibition of site IQ ROS partially improves muscle regeneration in adult but not old muscle demonstrating a role for CI ROS in the response to muscle injury. Site IQ ROS does not contribute to impaired regenerative capacity in aging.

Deletion of SA ß-Gal+ cells using senolytics improves muscle regeneration in old mice.

Systemic deletion of senescent cells leads to robust improvements in cognitive, cardiovascular, and whole-body metabolism, but their role in tissue reparative processes is incompletely understood. We hypothesized that senolytic drugs would enhance regeneration in aged skeletal muscle. Young (3 months) and old (20 months) male C57Bl/6J mice were administered the senolytics dasatinib (5 mg/kg) and quercetin (50 mg/kg) or vehicle bi-weekly for 4 months. Tibialis anterior (TA) was then injected with 1.2% BaCl2 or PBS 7- or 28 days prior to euthanization. Senescence-associated ß-Galactosidase positive (SA ß-Gal+) cell abundance was low in muscle from both young and old mice and increased similarly 7 days following injury in both age groups, with no effect of D+Q. Most SA ß-Gal+ cells were also CD11b+ in young and old mice 7- and 14 days following injury, suggesting they are infiltrating immune cells. By 14 days, SA ß-Gal+/CD11b+ cells from old mice expressed senescence genes, whereas those from young mice expressed higher levels of genes characteristic of anti-inflammatory macrophages. SA ß-Gal+ cells remained elevated in old compared to young mice 28 days following injury, which were reduced by D+Q only in the old mice. In D+Q-treated old mice, muscle regenerated following injury to a greater extent compared to vehicle-treated old mice, having larger fiber cross-sectional area after 28 days. Conversely, D+Q blunted regeneration in young mice. In vitro experiments suggested D+Q directly improve myogenic progenitor cell proliferation. Enhanced physical function and improved muscle regeneration demonstrate that senolytics have beneficial effects only in old mice.

The biphasic and age-dependent impact of klotho on hallmarks of aging and skeletal muscle function.

Aging is accompanied by disrupted information flow, resulting from accumulation of molecular mistakes. These mistakes ultimately give rise to debilitating disorders including skeletal muscle wasting, or sarcopenia. To derive a global metric of growing 'disorderliness' of aging muscle, we employed a statistical physics approach to estimate the state parameter, entropy, as a function of genes associated with hallmarks of aging. Escalating network entropy reached an inflection point at old age, while structural and functional alterations progressed into oldest-old age. To probe the potential for restoration of molecular 'order' and reversal of the sarcopenic phenotype, we systemically overexpressed the longevity protein, Klotho, via AAV. Klotho overexpression modulated genes representing all hallmarks of aging in old and oldest-old mice, but pathway enrichment revealed directions of changes were, for many genes, age-dependent. Functional improvements were also age-dependent. Klotho improved strength in old mice, but failed to induce benefits beyond the entropic tipping point.

Characterizing pancreatic ß-cell heterogeneity in the streptozotocin model by single-cell transcriptomic analysis.

OBJECTIVES: The streptozotocin (STZ) model is widely used in diabetes research. However, the cellular and molecular states of pancreatic endocrine cells in this model remain unclear. This study explored the molecular characteristics of islet cells treated with STZ and re-evaluated ß-cell dysfunction and regeneration in the STZ model. METHODS: We performed single-cell RNA sequencing of pancreatic endocrine cells from STZ-treated mice. High-quality sequencing data from 2,999 cells were used to identify clusters via Louvain clustering analysis. Principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), uniform manifold approximation and projection (UMAP), force-directed layout (FDL), and differential expression analysis were used to define the heterogeneity and transcriptomic changes in islet cells. In addition, qPCR and immunofluorescence staining were used to confirm findings from the sequencing data. RESULTS: Untreated ß-cells were divided into two populations at the transcriptomic level, a large high-Glut2 expression (Glut2high) population and a small low-Glut2 expression (Glut2low) population. At the transcriptomic level, Glut2low ß-cells in adult mice did not represent a developmentally immature state, although a fraction of genes associated with ß-cell maturation and function were downregulated in Glut2low cells. After a single high-dose STZ treatment, most Glut2high cells were killed, but Glut2low cells survived and over time changed to a distinct cell state. We did not observe conversion of Glut2low to Glut2high ß-cells up to 9 months after STZ treatment. In addition, we did not detect transcriptomic changes in the non-ß endocrine cells or a direct trans-differentiation pathway from the α-cell lineage to the ß-cell lineage in the STZ model. CONCLUSIONS: We identified the heterogeneity of ß-cells in both physiological and pathological conditions. However, we did not observe conversion of Glut2low to Glut2high ß-cells, transcriptomic changes in the non-ß endocrine cells, or direct trans-differentiation from the α-cell lineage to the ß-cell lineage in the STZ model. Our results clearly define the states of islet cells treated with STZ and allow us to re-evaluate the STZ model widely used in diabetes studies.

Klotho: An Elephant in Aging Research.

The year 2017 marked the 20th anniversary of the first publication describing Klotho. This single protein was and is remarkable in that its absence in mice conferred an accelerated aging, or progeroid, phenotype with a dramatically shortened life span. On the other hand, genetic overexpression extended both health span and life span by an impressive 30%. Not only has Klotho deficiency been linked to a number of debilitating age-related illnesses but many subsequent reports have lent credence to the idea that Klotho can compress the period of morbidity and extend the life span of both model organisms and humans. This suggests that Klotho functions as an integrator of organ systems, making it both a promising tool for advancing our understanding of the biology of aging and an intriguing target for interventional studies. In this review, we highlight advances in our understanding of Klotho as well as key challenges that have somewhat limited our view, and thus translational potential, of this potent protein.

GDF11 Decreases Pressure Overload-Induced Hypertrophy, but Can Cause Severe Cachexia and Premature Death.

RATIONALE: Possible beneficial effects of GDF11 (growth differentiation factor 11) on the normal, diseased, and aging heart have been reported, including reversing aging-induced hypertrophy. These effects have not been well validated. High levels of GDF11 have also been shown to cause cardiac and skeletal muscle wasting. These controversies could be resolved if dose-dependent effects of GDF11 were defined in normal and aged animals as well as in pressure overload-induced pathological hypertrophy. OBJECTIVE: To determine dose-dependent effects of GDF11 on normal hearts and those with pressure overload-induced cardiac hypertrophy. METHODS AND RESULTS: Twelve- to 13-week-old C57BL/6 mice underwent transverse aortic constriction (TAC) surgery. One-week post-TAC, these mice received rGDF11 (recombinant GDF11) at 1 of 3 doses: 0.5, 1.0, or 5.0 mg/kg for up to 14 days. Treatment with GDF11 increased plasma concentrations of GDF11 and p-SMAD2 in the heart. There were no significant differences in the peak pressure gradients across the aortic constriction between treatment groups at 1 week post-TAC. Two weeks of GDF11 treatment caused dose-dependent decreases in cardiac hypertrophy as measured by heart weight/tibia length ratio, myocyte cross-sectional area, and left ventricular mass. GDF11 improved cardiac pump function while preventing TAC-induced ventricular dilation and caused a dose-dependent decrease in interstitial fibrosis (in vivo), despite increasing markers of fibroblast activation and myofibroblast transdifferentiation (in vitro). Treatment with the highest dose (5.0 mg/kg) of GDF11 caused severe body weight loss, with significant decreases in both muscle and organ weights and death in both sham and TAC mice. CONCLUSIONS: Although GDF11 treatment can reduce pathological cardiac hypertrophy and associated fibrosis while improving cardiac pump function in pressure overload, high doses of GDF11 cause severe cachexia and death. Use of GDF11 as a therapy could have potentially devastating actions on the heart and other tissues.

GDF11 Treatment Attenuates the Recovery of Skeletal Muscle Function After Injury in Older Rats.

Loss of skeletal muscle mass and function results in loss of mobility for elderly patients. Novel therapies that can protect and/or restore muscle function during aging would have profound effects on the quality of life for this population. Growth differentiation factor 11 (GDF11) has been proposed as a "youthful" circulating factor that can restore cardiac, neural, and skeletal muscle functions in aging animals. However, conflicting data has been recently published that casts doubt on these assertions. We used a complex rat model of skeletal muscle injury that physiologically mimics injuries seen in patients; to investigate the ability of GDF11 and to enhance skeletal muscle regeneration after injury in older rats. Our data showed that GDF11 treatment resulted in a significant increase in tissue fibrosis, accompanied by attenuated functional recovery, as compared to animals treated with vehicle alone. GDF11 impaired the recovery of skeletal muscle function in older rats after injury.

High-Throughput Small Interfering RNA Screening Identifies Phosphatidylinositol 3-Kinase Class II Alpha as Important for Production of Human Cytomegalovirus Virions.

UNLABELLED: High-throughput small interfering RNA (siRNA) screening is a useful methodology to identify cellular factors required for virus replication. Here we utilized a high-throughput siRNA screen based on detection of a viral antigen by microscopy to interrogate cellular protein kinases and phosphatases for their importance during human cytomegalovirus (HCMV) replication and identified the class II phosphatidylinositol 3-kinase class II alpha (PI3K-C2A) as being involved in HCMV replication. Confirming this observation, infected cells treated with either pooled or individual siRNAs targeting PI3K-C2A mRNA produced approximately 10-fold less infectious virus than the controls. Western blotting and quantitative PCR analysis of infected cells treated with siRNAs indicated that depletion of PI3K-C2A slightly reduced the accumulation of late but not immediate early or early viral antigens and had no appreciable effect on viral DNA synthesis. Analysis of siRNA-treated cells by electron microscopy and Western blotting indicated that PI3K-C2A was not required for the production of viral capsids but did lead to increased numbers of enveloped capsids in the cytoplasm that had undergone secondary envelopment and a reduction in the amount of viral particles exiting the cell. Therefore, PI3K-C2A is a factor important for HCMV replication and has a role in the production of HCMV virions. IMPORTANCE: There is limited information about the cellular factors required for human cytomegalovirus (HCMV) replication. Therefore, to identify proteins involved in HCMV replication, we developed a methodology to conduct a high-throughput siRNA screen of HCMV-infected cells. From our screening data, we focused our studies on the top hit from our screen, the lipid kinase phosphatidylinositol 3-kinase class II alpha (PI3K-C2A), as its role in HCMV replication was unknown. Interestingly, we found that PI3K-C2A is important for the production of HCMV virions and is involved in virion production after secondary envelopment of viral capsids, the encapsidation of HCMV capsids by a lipid bilayer that occurs before virions exit the cell.

Differential Binding Activity of TGF-ß Family Proteins to Select TGF-ß Receptors.

Growth differentiation factor-11 (GDF11) and myostatin (MSTN) are highly related transforming growth factor-ß (TGF-ß) ligands with 89% amino acid sequence homology. They have different biologic activities and diverse tissue distribution patterns. However, the activities of these ligands are indistinguishable in in vitro assays. SMAD2/3 signaling has been identified as the canonical pathway for GDF11 and MSTN, However, it remains unclear which receptor heterodimer and which antagonists preferentially mediate and regulate signaling. In this study, we investigated the initiation and regulation of GDF11 and MSTN signaling at the receptor level using a novel receptor dimerization detection technology. We used the dimerization platform to link early receptor binding events to intracellular downstream signaling. This approach was instrumental in revealing differential receptor binding activity within the TGF-ß family. We verified the ActR2b/ALK5 heterodimer as the predominant receptor for GDF11- and MSTN-induced SMAD2/3 signaling. We also showed ALK7 specifically mediates activin-B signaling. We verified follistatin as a potent antagonist to neutralize both SMAD2/3 signaling and receptor dimerization. More remarkably, we showed that the two related antagonists, growth and differentiation factor-associated serum protein (GASP)-1 and GASP2, differentially regulate GDF11 (and MSTN) signaling. GASP1 blocks both receptor dimerization and downstream signaling. However, GASP2 blocks only downstream signaling without interference from receptor dimerization. Our data strongly suggest that physical binding of GDF11 (and MSTN) to both ActR2b and ALK5 receptors is required for initiation of signaling.

Discovery of Potent, Orally Bioavailable Inhibitors of Human Cytomegalovirus.

A high-throughput screen based on a viral replication assay was used to identify inhibitors of the human cytomegalovirus. Using this approach, hit compound 1 was identified as a 4 µM inhibitor of HCMV that was specific and selective over other herpes viruses. Time of addition studies indicated compound 1 exerted its antiviral effect early in the viral life cycle. Mechanism of action studies also revealed that this series inhibited infection of MRC-5 and ARPE19 cells by free virus and via direct cell-to-cell spread from infected to uninfected cells. Preliminary structure-activity relationships demonstrated that the potency of compound 1 could be improved to a low nanomolar level, but metabolic stability was a key optimization parameter for this series. A strategy focused on minimizing metabolic hydrolysis of the N1-amide led to an alternative scaffold in this series with improved metabolic stability and good pharmacokinetic parameters in rat.

Is Growth Differentiation Factor 11 a Realistic Therapeutic for Aging-Dependent Muscle Defects?

This "Controversies in Cardiovascular Research" article evaluates the evidence for and against the hypothesis that the circulating blood level of growth differentiation factor 11 (GDF11) decreases in old age and that restoring normal GDF11 levels in old animals rejuvenates their skeletal muscle and reverses pathological cardiac hypertrophy and cardiac dysfunction. Studies supporting the original GDF11 hypothesis in skeletal and cardiac muscle have not been validated by several independent groups. These new studies have either found no effects of restoring normal GDF11 levels on cardiac structure and function or have shown that increasing GDF11 or its closely related family member growth differentiation factor 8 actually impairs skeletal muscle repair in old animals. One possible explanation for what seems to be mutually exclusive findings is that the original reagent used to measure GDF11 levels also detected many other molecules so that age-dependent changes in GDF11 are still not well known. The more important issue is whether increasing blood [GDF11] repairs old skeletal muscle and reverses age-related cardiac pathologies. There are substantial new and existing data showing that GDF8/11 can exacerbate rather than rejuvenate skeletal muscle injury in old animals. There is also new evidence disputing the idea that there is pathological hypertrophy in old C57bl6 mice and that GDF11 therapy can reverse cardiac pathologies. Finally, high [GDF11] causes reductions in body and heart weight in both young and old animals, suggestive of a cachexia effect. Our conclusion is that elevating blood levels of GDF11 in the aged might cause more harm than good.

Crystal structure of human GDF11.

Members of the TGF-ß family of proteins are believed to play critical roles in cellular signaling processes such as those involved in muscle differentiation. The extent to which individual family members have been characterized and linked to biological function varies greatly. The role of myostatin, also known as growth differentiation factor 8 (GDF8), as an inhibitor of muscle differentiation is well understood through genetic linkages. In contrast, the role of growth differentiation factor 11 (GDF11) is much less well understood. In humans, the mature forms of GDF11 and myostatin are over 94% identical. In order to understand the role that the small differences in sequence may play in the differential signaling of these molecules, the crystal structure of GDF11 was determined to a resolution of 1.50 Å. A comparison of the GDF11 structure with those of other family members reveals that the canonical TGF-ß domain fold is conserved. A detailed structural comparison of GDF11 and myostatin shows that several of the differences between these proteins are likely to be localized at interfaces that are critical for the interaction with downstream receptors and inhibitors.

Development of an ultra-sensitive Simoa assay to enable GDF11 detection: a comparison across bioanalytical platforms.

BACKGROUND: Four bioanalytical platforms were evaluated to optimize sensitivity and enable detection of recombinant human GDF11 in biological matrices; ELISA, Meso Scale Discovery, Gyrolab xP Workstation and Simoa HD-1. Results & methodology: After completion of custom assay development, the single-molecule ELISA (Simoa) achieved the greatest sensitivity with a lower limit of quantitation of 0.1 ng/ml, an improvement of 100-fold over the next sensitive platform (MSD). DISCUSSION & CONCLUSION: This improvement was essential to enable detection of GDF11 in biological samples, and without the technology the sensitivity achieved on the other platforms would not have been sufficient. Other factors such as ease of use, cost, assay time and automation capability can also be considered when developing custom immunoassays, based on the requirements of the bioanalyst.

GDF11 does not rescue aging-related pathological hypertrophy.

RATIONALE: Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor-ß super family of secreted factors. A recent study showed that reduced GDF11 blood levels with aging was associated with pathological cardiac hypertrophy (PCH) and restoring GDF11 to normal levels in old mice rescued PCH. OBJECTIVE: To determine whether and by what mechanism GDF11 rescues aging dependent PCH. METHODS AND RESULTS: Twenty-four-month-old C57BL/6 mice were given a daily injection of either recombinant (r) GDF11 at 0.1 mg/kg or vehicle for 28 days. rGDF11 bioactivity was confirmed in vitro. After treatment, rGDF11 levels were significantly increased, but there was no significant effect on either heart weight or body weight. Heart weight/body weight ratios of old mice were not different from 8- or 12-week-old animals, and the PCH marker atrial natriuretic peptide was not different in young versus old mice. Ejection fraction, internal ventricular dimension, and septal wall thickness were not significantly different between rGDF11 and vehicle-treated animals at baseline and remained unchanged at 1, 2, and 4 weeks of treatment. There was no difference in myocyte cross-sectional area rGDF11 versus vehicle-treated old animals. In vitro studies using phenylephrine-treated neonatal rat ventricular myocytes, to explore the putative antihypertrophic effects of GDF11, showed that GDF11 did not reduce neonatal rat ventricular myocytes hypertrophy, but instead induced hypertrophy. CONCLUSIONS: Our studies show that there is no age-related PCH in disease-free 24-month-old C57BL/6 mice and that restoring GDF11 in old mice has no effect on cardiac structure or function.

RNASEK Is a V-ATPase-Associated Factor Required for Endocytosis and the Replication of Rhinovirus, Influenza A Virus, and Dengue Virus.

Human rhinovirus (HRV) causes upper respiratory infections and asthma exacerbations. We screened multiple orthologous RNAi reagents and identified host proteins that modulate HRV replication. Here, we show that RNASEK, a transmembrane protein, was needed for the replication of HRV, influenza A virus, and dengue virus. RNASEK localizes to the cell surface and endosomal pathway and closely associates with the vacuolar ATPase (V-ATPase) proton pump. RNASEK is required for endocytosis, and its depletion produces enlarged clathrin-coated pits (CCPs) at the cell surface. These enlarged CCPs contain endocytic cargo and are bound by the scissioning GTPase, DNM2. Loss of RNASEK alters the localization of multiple V-ATPase subunits and lowers the levels of the ATP6AP1 subunit. Together, our results show that RNASEK closely associates with the V-ATPase and is required for its function; its loss prevents the early events of endocytosis and the replication of multiple pathogenic viruses.

Development of a high-throughput human cytomegalovirus quantitative PCR cell-based assay.

This report describes the development and optimization of a quantitative real-time PCR assay for evaluating human cytomegalovirus (CMV) replication in vitro and susceptibility to antiviral drugs. This assay measures the level of intracellular CMV DNA in both 96- and 384-well microplate formats. Normalization of CMV levels using mitochondrial DNA enhanced the robustness of the assay and minimized variability. The assay throughput was further enhanced by eliminating several wash steps and by lysing the cells directly in the presence of cell culture media, both of which had no impact on the assay metrics. The assay was validated using several known CMV antiviral compounds. The CMV quantitative PCR (qPCR) assay represents a rapid, reliable and reproducible method that can be used with both CMV laboratory strains and clinical isolates.

Neutralizing antibodies are unable to inhibit direct viral cell-to-cell spread of human cytomegalovirus.

Infection with human cytomegalovirus (CMV) during pregnancy is the most common cause of congenital disorders, and can lead to severe life-long disabilities with associated high cost of care. Since there is no vaccine or effective treatment, current efforts are focused on identifying potent neutralizing antibodies. A panel of CMV monoclonal antibodies identified from patent applications, was synthesized and expressed in order to reproduce data from the literature showing that anti-glycoprotein B antibodies neutralized virus entry into all cell types and that anti-pentameric complex antibodies are highly potent in preventing virus entry into epithelial cells. It had not been established whether antibodies could prevent subsequent rounds of infection that are mediated primarily by direct cell-to-cell transmission. A thorough validation of a plaque reduction assay to monitor cell-to-cell spread led to the conclusion that neutralizing antibodies do not significantly inhibit plaque formation or reduce plaque size when they are added post-infection.

Phosphatidylinositol 4-kinase III beta is essential for replication of human rhinovirus and its inhibition causes a lethal phenotype in vivo.

Human rhinovirus (HRV) is the predominant cause of the common cold, but more importantly, infection may have serious repercussions in asthmatics and chronic obstructive pulmonary disorder (COPD) patients. A cell-based antiviral screen against HRV was performed with a subset of our proprietary compound collection, and an aminothiazole series with pan-HRV species and enteroviral activity was identified. The series was found to act at the level of replication in the HRV infectious cycle. In vitro selection and sequencing of aminothiazole series-resistant HRV variants revealed a single-nucleotide mutation leading to the amino acid change I42V in the essential HRV 3A protein. This same mutation has been previously implicated in resistance to enviroxime, a former clinical-stage antipicornavirus agent. Enviroxime-like compounds have recently been shown to target the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIß). A good correlation between PI4KIIIß activity and HRV antiviral potency was found when analyzing the data over 80 compounds of the aminothiazole series, covering a 750-fold potency range. The mechanism of action through PI4KIIIß inhibition was further demonstrated by small interfering RNA (siRNA) knockdown of PI4KB, which reduced HRV replication and also increased the potency of the PI4KIIIß inhibitors. Inhibitors from two different structural classes with promising pharmacokinetic profiles and with very good selectivity for PI4KIIIß were used to dissociate compound-related toxicity from target-related toxicity. Mortality was seen in all dosing groups of mice treated with either compound, therefore suggesting that short-term inhibition of PI4KIIIß is deleterious.

Vectored co-delivery of human cytomegalovirus gH and gL proteins elicits potent complement-independent neutralizing antibodies.

Human cytomegalovirus (hCMV) is prevalent worldwide with infection generally being asymptomatic. Nevertheless, hCMV infection can lead to significant morbidity and mortality. Primary infection of seronegative women or reactivation/re-infection of seropositive women during pregnancy can result in transmission to the fetus, leading to severe neurological defects. In addition, hCMV is the most common viral infection in immunosuppressed organ transplant recipients and can produce serious complications. Hence, a safe and effective vaccine to prevent hCMV infection is an unmet medical need. Neutralizing antibodies to several hCMV glycoproteins, and complexes thereof, have been identified in individuals following hCMV infection. Interestingly, a portion of the CMV-specific neutralizing antibody responses are directed to epitopes found on glycoprotein complexes but not the individual proteins. Using an alphavirus replicon particle (VRP) vaccine platform, we showed that bicistronic VRPs encoding hCMV gH and gL glycoproteins produce gH/gL complexes in vitro. Furthermore, mice vaccinated with these gH/gL-expressing VRPs produced broadly cross-reactive complement-independent neutralizing antibodies to hCMV. These neutralizing antibody responses were of higher titer than those elicited in mice vaccinated with monocistronic VRPs encoding gH or gL antigens, and they were substantially more potent than those raised by VRPs encoding gB. These findings underscore the utility of co-delivery of glycoprotein components such as gH and gL for eliciting potent, broadly neutralizing immune responses against hCMV, and indicate that the gH/gL complex represents a potential target for future hCMV vaccine development.