Sickle cell disease (SCD) is an autosomal recessive disorder of blood characterized by a mutation in the β chain of hemoglobin (Hb), leading to the production of sickle Hb (HbS). In SCD, under low oxygen conditions, red blood cells (RBCs) containing HbS form a characteristic "sickle" shape, resulting in chronic hemolytic anemia and acute vaso-occlusive crises. Current therapies for SCD have limitations in efficacy or availability, highlighting the need for new anti-sickling drugs. To facilitate the discovery of new anti-sickling compounds, we previously developed a high throughput sickling assay, which permits rapid screening of thousands of compounds for the ability to inhibit RBC sickling. In this study, we improved the sickling assay by optimizing the assay condition and expanded our screening efforts by evaluating the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) compound library, which contains approximately 2.5 times more compounds than previously screened. We were able to increase the number of blood samples that were adequate for identifying anti-sickling compounds in the improved sickling assay and identified voxelotor and SNS-314 as compounds that successfully prevented sickling. The improved sickling assay will increase access to valuable blood samples from SCD volunteers, providing more opportunities to develop anti-sickling compounds for treating SCD.

Respiratory syncytial virus (RSV) is a common cause of acute lower respiratory tract infection in infants, older adults and the immunocompromised. Effective directly acting antivirals are not yet available for clinical use. To address this, we screen the ReFRAME drug-repurposing library consisting of 12,000 small molecules against RSV. We identify 21 primary candidates including RSV F and N protein inhibitors, five HSP90 and four IMPDH inhibitors. We select lonafarnib, a licensed farnesyltransferase inhibitor, and phase III candidate for hepatitis delta virus (HDV) therapy, for further follow-up. Dose-response analyses and plaque assays confirm the antiviral activity (IC 50 : 10-118 nM). Passaging of RSV with lonafarnib selects for phenotypic resistance and fixation of mutations in the RSV fusion protein (T335I and T400A). Lentiviral pseudotypes programmed with variant RSV fusion proteins confirm that lonafarnib inhibits RSV cell entry and that these mutations confer lonafarnib resistance. Surface plasmon resonance reveals RSV fusion protein binding of lonafarnib and co-crystallography identifies the lonafarnib binding site within RSV F. Oral administration of lonafarnib dose-dependently reduces RSV virus load in a murine infection model using female mice. Collectively, this work provides an overview of RSV drug repurposing candidates and establishes lonafarnib as a bona fide fusion protein inhibitor.

Osteoarthritis (OA) is a common, debilitating, chronic disease with no disease-modifying drug approved to date. We discovered LNA043—a derivative of angiopoietin-like 3 (ANGPTL3)—as a potent chondrogenesis inducer using a phenotypic screen with human mesenchymal stem cells. We show that LNA043 promotes chondrogenesis and cartilage matrix synthesis in vitro and regenerates hyaline articular cartilage in preclinical OA and cartilage injury models in vivo. LNA043 exerts at least part of these effects through binding to the fibronectin receptor, integrin α 5 β 1 on mesenchymal stem cells and chondrocytes. In a first-in-human (phase 1), randomized, double-blinded, placebo-controlled, single ascending dose, single-center trial ( NCT02491281 ; sponsored by Novartis Pharmaceuticals), 28 patients with knee OA were injected intra-articularly with LNA043 or placebo (3:1 ratio) either 2 h, 7 d or 21 d before total knee replacement. LNA043 met its primary safety endpoint and showed short serum pharmacokinetics, cartilage penetration and a lack of immunogenicity (secondary endpoints). Post-hoc transcriptomics profiling of cartilage revealed that a single LNA043 injection reverses the OA transcriptome signature over at least 21 d, inducing the expression of hyaline cartilage matrix components and anabolic signaling pathways, while suppressing mediators of OA progression. LNA043 is a novel disease-modifying OA drug candidate that is currently in a phase 2b trial ( NCT04864392 ) in patients with knee OA.

Purpose: The goal of this study was to identify a disease modifying osteoarthritis drug (DMOAD) that regenerates healthy hyaline cartilage in osteoarthritis (OA) through differentiation of endogenous mesenchymal stem cells (MSCs) into chondrocytes and the production of healthy hyaline cartilage matrix. Using a phenotypic screen, we discovered LNA043, a derivative of the angiopoietin-like 3 (ANGPTL3) protein and confirmed that it promotes chondrogenesis in vitro and cartilage regeneration in vivo.

Purpose: Type 1 diabetes (T1D) is a disease affecting almost three million Americans; every year 30,000 new cases are diagnosed, 50% of whom are children. There is currently no curative therapy for T1D and the only available treatment is insulin replacement. Substantial recent data demonstrate a strong association between the MHC Class II molecule DQ8 and the development of T1D. Indeed, DQ8 has been shown to present antigenic peptides (such as InsB:9-23 and GAD65) driving activation of CD4+ T cells in T1D patients. The goal of this study was to identify and optimize a monoclonal antibody that binds to DQ8 molecule and blocks the continued activation of DQ8 restricted T cells. Methods: Hybridoma clones were generated from spleens of BALB/c mice immunized with DQ8 molecule bound with the InsB:9-23 peptide and screened by high throughput flow cytometry using human B-cells expressing DQ8. 28 clones found positive by flow cytometry were functionally tested in a mixed lymphocyte reaction containing a murine T cell line expressing a human TCR specific for the InsB: 9-23 - DQ8 complex. DQ8-103, a murine version of our best candidate antibody (DQ8-27), was tested in transgenic DQ8 mice immunized with GAD65 peptide; Cytokine production from the recall response ex vivo and in vivo was evaluated using a Luminex assay. Immunophenotyping of blood, spleens and draining lymph nodes isolated from immunized DQ8 mice was done by mass cytometry (CyTOF). Results: We generated a monoclonal antibody against DQ8 (DQ8-27), that was able to bind human DQ8 molecule expressed on human B-cells and on murine splenocytes isolated from transgenic DQ8 mice. The murine version of DQ8-27 (DQ8-103) was able to significantly inhibit T cell activation in DQ8 mice immunized with GAD65 both ex vivo and in vivo, by decreasing the production of pro inflammatory cytokines IL-2 and IFN-γ. Of note, DQ8-103 caused a significant depletion of DQ8+ B-cells in treated mice compared to mice injected with a control antibody. Disclosure A. Lombardi: None. M. Zhu: None. T. Moran: None. T.A. Kraus: None. M. Hsieh: None. C. Stevens: None. D. Murphy: None. C.B. Donovan: Employee; Self; Pfizer Inc.. K. Atkuri: None. D.M. Messing: None. J. Chamoun: None. T.P. Brown: None. G. Guntas: None. D.V. Bennett: None. E. Concepcion: None. K. Johnson: None. Y. Tomer: None.

Fibroblast growth factor 23 (FGF23) is the causative factor of X-linked hypophosphatemia (XLH), a genetic disorder effecting 1:20,000 that is characterized by excessive phosphate excretion, elevated FGF23 levels and a rickets/osteomalacia phenotype. FGF23 inhibits phosphate reabsorption and suppresses 1α,25-dihydroxyvitamin D (1,25D) biosynthesis, analytes that differentially contribute to bone integrity and deleterious soft-tissue mineralization. As inhibition of ligand broadly modulates downstream targets, balancing efficacy and unwanted toxicity is difficult when targeting the FGF23 pathway. We demonstrate that a FGF23 c-tail-Fc fusion molecule selectively modulates the phosphate pathway in vivo by competitive antagonism of FGF23 binding to the FGFR/α klotho receptor complex. Repeated injection of FGF23 c-tail Fc in Hyp mice, a preclinical model of XLH, increases cell surface abundance of kidney NaPi transporters, normalizes phosphate excretion, and significantly improves bone architecture in the absence of soft-tissue mineralization. Repeated injection does not modulate either 1,25D or calcium in a physiologically relevant manner in either a wild-type or disease setting. These data suggest that bone integrity can be improved in models of XLH via the exclusive modulation of phosphate. We posit that the selective modulation of the phosphate pathway will increase the window between efficacy and safety risks, allowing increased efficacy to be achieved in the treatment of this chronic disease. © 2017 American Society for Bone and Mineral Research.

Significance There is considerable interest in developing immunosuppressants that can specifically target effector memory T cells which are key to the pathogenesis of many inflammatory disorders. The potassium channel Kv1.3 has been found to play an important role in T EM activation, but not in naive and central memory T cells. It has proven challenging to generate small molecules or antibodies that potently and selectively block Kv1.3 function. We generated antibody fusions by grafting potent Kv1.3 blocking peptides into complementary-determining regions (CDRs) of humanized antibodies, and among those, identified a candidate with high in vitro potency, excellent physiochemical and pharmacologic properties, and in vivo efficacy in a rat model. These novel toxin-antibody fusions have therapeutic potential for a variety of T EM -mediated inflammatory diseases.

The recent study by Miller et al. (1) provides a comprehensive, quantitative analysis of anthropogenic methane sources in the United States using atmospheric methane observations, spatial datasets, and a high-resolution atmospheric transport model. The authors conclude that “…emissions due to ruminants and manure are up to twice the magnitude of existing [i.e., US Environmental Protection Agency (US EPA); www.epa.gov/climatechange/ghgemissions/usinventoryreport.html] inventories” (1). The validity of this “top-down” approach can be verified by a relatively simple “bottom-up” method using current livestock inventories and enteric or manure methane emission factors. Animal scientists have generated large datasets of enteric methane production estimates per unit of feed or energy intake. Methanogenesis in the rumen is substrate-dependent and methane production data derived from studies using respiratory chambers (or other techniques) expressed on feed intake basis are representative of field emissions, if feed intake is known. We used the US Department of Agriculture-National Agricultural Statistics Service (USDA-NASS) livestock inventory estimates for 2013 (www.nass.usda.gov) and methane emission rates per unit of feed dry matter intake from two large datasets [Hristov et al. (2) and Hales et al. (3)] to estimate total methane emission from enteric fermentation for the United States. Total cattle inventories for 2013 were 89,299,600 head (including 29,295,200 beef cows, 9,219,900 dairy cows, and 13,351,700 cattle on feed, among other categories). Feed dry matter intake was estimated based on beef and dairy cattle requirements and ranged from 3.8 (calves 500 lbs), 11 (beef cows), and 22 kg/d (dairy cows). Methane production rates were estimated at 8–13 (cattle on feed) or 20 g/kg (all other categories; SD = 4) feed dry matter intake. Contributions to methane emissions by other ruminants or nonruminant herbivores (sheep, goats, wild ruminants, horses, and so forth) are small in the United States and were not included in this analysis. With the above assumptions, total methane emissions from enteric fermentation were estimated at 6.241 Tg/yr (minimum = 4.972 and maximum = 7.511), which is comparable to the current, 2011 US EPA estimates of 6.542 Tg/yr and was also independently verified using equations proposed by Moraes et al. (4). USDA-NASS inventories for cattle, swine (59,387,000 market swine and 5,834,000 breeding swine), and poultry (a total of 8.562 billion birds) and Intergovernmental Panel on Climate Change (5) manure methane emission factors [from 0.02 (most poultry categories), to 1 (beef cattle) and 53 (dairy cows) kilograms per head per year] were used to estimate emissions from manure management. Using this approach, manure emissions in the United States were estimated at 1.604 Tg/yr, which is lower than the 2011 US EPA estimate of 2.478 Tg/yr (with the latter figure perhaps being more representative of manure systems in the United States). Thus, the conclusions by Miller et al. (1) that US EPA estimates for livestock methane emissions are grossly underestimated appears to be unsubstantiated by the above “bottom-up” approach. There is a need for a detailed inventory of manure systems for all farm animal species and categories, which will help to more accurately estimate greenhouse gas (and ammonia) emissions from animal manure in the United States.

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Osteoarthritis (OA) is a degenerative joint disease that involves the destruction of articular cartilage and eventually leads to disability. Molecules that promote the selective differentiation of multipotent mesenchymal stem cells (MSCs) into chondrocytes may stimulate the repair of damaged cartilage. Using an image-based high-throughput screen, we identified the small molecule kartogenin, which promotes chondrocyte differentiation (median effective concentration = 100 nM), shows chondroprotective effects in vitro, and is efficacious in two OA animal models. Kartogenin binds filamin A, disrupts its interaction with the transcription factor core-binding factor β subunit (CBFβ), and induces chondrogenesis by regulating the CBFβ-RUNX1 transcriptional program. This work provides new insights into the control of chondrogenesis that may ultimately lead to a stem cell-based therapy for osteoarthritis.

Regeneration of peripheral differentiated tissue in mammals is rare, and regulators of this process are largely unknown. We carried out a forward genetic screen in mice using N -ethyl- N -nitrosourea mutagenesis to identify genetic mutations that affect regenerative healing in vivo. More than 400 pedigrees were screened for closure of a through-and-through punch wound in the mouse ear. This led to the identification of a single pedigree with a heritable, fast, and regenerative wound-healing phenotype. Within 5 wk after ear-punch, a threefold decrease in the diameter of the wound was observed in the mutant mice compared with the wild-type mice. At 22 wk, new cartilage, hair follicles, and sebaceous glands were observed in the newly generated tissue. This trait was mapped to a point mutation in a receptor for TGF-β, TGFBR1 . Mouse embryonic fibroblasts from the affected mice had increased expression of a subset of TGF-β target genes, suggesting that the mutation caused partial activation of the receptor. Further, bone marrow stromal cells from the mutant mice more readily differentiated to chondrogenic precursors, providing a plausible explanation for the enhanced development of cartilage islands in the regenerated ears. This mutant mouse strain provides a unique model to further explore regeneration in mammals and, in particular, the role of TGFBR1 in chondrogenesis and regenerative wound healing.

Background: Insulin resistance is manifested in muscle, adipose tissue, and liver and is associated with adipose tissue inflammation. The cellular components and mechanisms that regulate the onset of diet-induced insulin resistance are not clearly defined.Methodology and Principal Findings: We initially observed osteopontin (OPN) mRNA over-expression in adipose tissue of obese, insulin resistant humans and rats which was normalized by thiazolidinedione (TZD) treatment in both species. OPN regulates inflammation and is implicated in pathogenic maladies resulting from chronic obesity. Thus, we tested the hypothesis that OPN is involved in the early development of insulin resistance using a 2-4 week high fat diet (HFD) model. OPN KO mice fed HFD for 2 weeks were completely protected from the severe skeletal muscle, liver and adipose tissue insulin resistance that developed in wild type (WT) controls, as determined by hyperinsulinemic euglycemic clamp and acute insulin-stimulation studies. Although two-week HFD did not alter body weight or plasma free fatty acids and cytokines in either strain, HFD-induced hyperleptinemia, increased adipose tissue inflammation (macrophages and cytokines), and adipocyte hypertrophy were significant in WT mice and blunted or absent in OPN KO mice. Adipose tissue OPN protein isoform expression was significantly altered in 2- and 4-week HFD-fed WT mice but total OPN protein was unchanged. OPN KO bone marrow stromal cells were more osteogenic and less adipogenic than WT cells in vitro. Interestingly, the two differentiation pathways were inversely affected by HFD in WT cells in vitro.Conclusions: The OPN KO phenotypes we report reflect protection from insulin resistance that is associated with changes in adipocyte biology and adipose tissue inflammatory status. OPN is a key component in the development of HFD-induced insulin resistance.

Approximately 3,500 mammalian genes are predicted to be secreted or single-pass transmembrane proteins. The function of the majority of these genes is still unknown, and a number of the encoded proteins might find use as new therapeutic agents themselves or as targets for small molecule or antibody drug development. To analyze the physiological activities of the extracellular proteome, we developed a large-scale, high-throughput protein expression, purification, and screening platform. For this study, the complete human extracellular proteome was analyzed and prioritized based on genome-wide disease association studies to select 529 initial target genes. These genes were cloned into three expression vectors as native sequences and as N-terminal and C-terminal Fc fusions to create an initial collection of 806 purified secreted proteins. To determine its utility, this library was screened in an OCT4-based cellular assay to identify regulators of human embryonic stem-cell self-renewal. We found that the pigment epithelium-derived factor can promote long-term pluripotent growth of human embryonic stem cells without bFGF or TGFβ/Activin/Nodal ligand supplementation. Our results further indicate that activation of the pigment epithelium-derived factor receptor-Erk1/2 signaling pathway by the pigment epithelium-derived factor is sufficient to maintain the self-renewal of pluripotent human embryonic stem cells. These experiments illustrate the potential for discovering novel biological functions by directly screening protein diversity in cell-based phenotypic or reporter assays.

ObjectiveThe transglutaminase (TG) isoenzyme TG2, which catalyzes protein cross-linking via transamidation, influences healing phenotype in multiple forms of tissue injury. Moreover, TG2 knockout suppresses cartilage destruction but promotes osteophyte formation in instability-induced mouse knee osteoarthritis (OA). TG2 is marker of growth plate chondrocyte hypertrophy. Moreover, TG2 secreted by chondrocytes acts in part by promoting chondrocyte maturation to hypertrophy, a differentiation state linked with MMP-13 expression and disease progression in OA. Moreover, glucosamine, which is currently under investigation as an OA therapy, binds and inhibits TG2. Here, we examined TG2 as a potential marker of cartilage hypertrophy in the spontaneous guinea pig model of OA.MethodsSynovial fluid ELISA and cartilage Immunohistochemistry and quantitative Reverse transcription-polymerase chain reaction (RT-PCR), were used to examine TG2 expression and TG transamidation-catalyzed isopeptide bonds.ResultsTG isopeptide bonds and TG2 were most abundant in articular cartilage in early knee OA. TG2 expression was robust at sites of early but not established osteophytes. Synovial fluid TG2 correlated with knee OA total histological score (r=0.47, P=0.01), as did medial tibial plateau cartilage TG2 mRNA (r=1.0, P=0.003). At 12 months of age, medial tibial plateau cartilage TG2 mRNA expression rose markedly in association with elevated type X collagen, as well as ADAMTS-5, and MMP-13 expression, changes not shared in age-matched Strain 13 guinea pigs that are less susceptible to knee OA.ConclusionHartley guinea pig knee TG2 expression associates with enhanced articular chondrocyte hypertrophy and is a biomarker of OA severity.

BACKGROUND:Health literacy screening is often not performed in clinical settings. One possible reason is the concern about the time involved in performing such assessments. Our objective was to measure the time required to administer the Newest Vital Sign (NVS) literacy assessment instrument to English-speaking primary care patients.METHODS:The NVS was administered to 78 consecutive English-speaking patients in an outpatient primary care clinic. The length of time to complete the NVS was timed with a stopwatch.RESULTS:The average time to complete the NVS was 2.9 minutes (95% confidence limit, 2.6-3.1 min).CONCLUSION:The NVS is a health literacy screening tool of sufficient brevity to be considered for use in primary care practices.