Clearance of plasma PCSK9 via the asialoglycoprotein receptor mediated by heterobifunctional ligands.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by promoting the degradation of hepatic LDL receptors (LDLRs). Current therapeutic approaches use antibodies that disrupt PCSK9 binding to LDLR to reduce circulating LDL-C concentrations or siRNA that reduces PCSK9 synthesis and thereby levels in circulation. Recent reports describe small molecules that, like therapeutic antibodies, interfere with PCSK9 binding to LDLR. We report an alternative approach to decrease circulating PCSK9 levels by accelerating PCSK9 clearance and degradation using heterobifunctional molecules that simultaneously bind to PCSK9 and the asialoglycoprotein receptor (ASGPR). Various formats, including bispecific antibodies, antibody-small molecule conjugates, and heterobifunctional small molecules, demonstrate binding in vitro and accelerated PCSK9 clearance in vivo. These molecules showcase a new approach to PCSK9 inhibition, targeted plasma protein degradation (TPPD), and demonstrate the feasibility of heterobifunctional small molecule ligands to accelerate the clearance and degradation of pathogenic proteins in circulation.

High-Throughput Screen for Inhibitors of Klebsiella pneumoniae Virulence Using a Tetrahymena pyriformis Co-Culture Surrogate Host Model.

The continuing emergence of antibacterial resistance reduces the effectiveness of antibiotics and drives an ongoing search for effective replacements. Screening compound libraries for antibacterial activity in standard growth media has been extensively explored and may be showing diminishing returns. Inhibition of bacterial targets that are selectively important under in vivo (infection) conditions and, therefore, would be missed by conventional in vitro screens might be an alternative. Surrogate host models of infection, however, are often not suitable for high-throughput screens. Here, we adapted a medium-throughput Tetrahymena pyriformis surrogate host model that was successfully used to identify inhibitors of a hyperviscous Klebsiella pneumoniae strain to a high-throughput format and screened circa 1.2 million compounds. The screen was robust and identified confirmed hits from different chemical classes with potent inhibition of K. pneumoniae growth in the presence of T. pyriformis that lacked any appreciable direct antibacterial activity. Several of these appeared to inhibit capsule/mucoidy, which are key virulence factors in hypervirulent K. pneumoniae. A weakly antibacterial inhibitor of LpxC (essential for the synthesis of the lipid A moiety of lipopolysaccharides) also appeared to be more active in the presence of T. pyriformis, which is consistent with the role of LPS in virulence as well as viability in K. pneumoniae.

Two RNA Tunnel Inhibitors Bind in Highly Conserved Sites in Dengue Virus NS5 Polymerase: Structural and Functional Studies.

Dengue virus (DENV) NS5 RNA-dependent RNA polymerase (RdRp), an important drug target, synthesizes viral RNA and is essential for viral replication. While a number of allosteric inhibitors have been reported for hepatitis C virus RdRp, few have been described for DENV RdRp. Following a diverse compound screening campaign and a rigorous hit-to-lead flowchart combining biochemical and biophysical approaches, two DENV RdRp nonnucleoside inhibitors were identified and characterized. These inhibitors show low- to high-micromolar inhibition in DENV RNA polymerization and cell-based assays. X-ray crystallography reveals that they bind in the enzyme RNA template tunnel. One compound (NITD-434) induced an allosteric pocket at the junction of the fingers and palm subdomains by displacing residue V603 in motif B. Binding of another compound (NITD-640) ordered the fingers loop preceding the F motif, close to the RNA template entrance. Most of the amino acid residues that interacted with these compounds are highly conserved in flaviviruses. Both sites are important for polymerase de novo initiation and elongation activities and essential for viral replication. This work provides evidence that the RNA tunnel in DENV RdRp offers interesting target sites for inhibition.IMPORTANCE Dengue virus (DENV), an important arthropod-transmitted human pathogen that causes a spectrum of diseases, has spread dramatically worldwide in recent years. Despite extensive efforts, the only commercial vaccine does not provide adequate protection to naive individuals. DENV NS5 polymerase is a promising drug target, as exemplified by the development of successful commercial drugs against hepatitis C virus (HCV) polymerase and HIV-1 reverse transcriptase. High-throughput screening of compound libraries against this enzyme enabled the discovery of inhibitors that induced binding sites in the RNA template channel. Characterizations by biochemical, biophysical, and reverse genetics approaches provide a better understanding of the biological relevance of these allosteric sites and the way forward to design more-potent inhibitors.

Advancements in Assay Technologies and Strategies to Enable Drug Discovery.

Assays drive drug discovery from the exploratory phases to the clinical testing of drug candidates. As such, numerous assay technologies and methodologies have arisen to support drug discovery efforts. Robust identification and characterization of tractable chemical matter requires biochemical, biophysical, and cellular approaches and often benefits from high-throughput methods. To increase throughput, efforts have been made to provide assays in miniaturized volumes which can be arrayed in microtiter plates to support the testing of as many as 100,000 samples/day. Alongside these efforts has been the growth of microtiter plate-free formats with encoded libraries that can support the screening of billions of compounds, a hunt for new drug modalities, as well as emphasis on more disease relevant formats using complex cell models of disease states. This review will focus on recent developments in high-throughput assay technologies applied to identify starting points for drug discovery. We also provide recommendations on strategies for implementing various assay types to select high quality leads for drug development.

Discovery of 4-((2S,4S)-4-Ethoxy-1-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)piperidin-2-yl)benzoic Acid (LNP023), a Factor B Inhibitor Specifically Designed To Be Applicable to Treating a Diverse Array of Complement Mediated Diseases.

The alternative pathway (AP) of the complement system is a key contributor to the pathogenesis of several human diseases including age-related macular degeneration, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and various glomerular diseases. The serine protease factor B (FB) is a key node in the AP and is integral to the formation of C3 and C5 convertase. Despite the prominent role of FB in the AP, selective orally bioavailable inhibitors, beyond our own efforts, have not been reported previously. Herein we describe in more detail our efforts to identify FB inhibitors by high-throughput screening (HTS) and leveraging insights from several X-ray cocrystal structures during optimization efforts. This work culminated in the discovery of LNP023 (41), which is currently being evaluated clinically in several diverse AP mediated indications.

A Phenotypic Screen Identifies Calcium Overload as a Key Mechanism of ß-Cell Glucolipotoxicity.

Type 2 diabetes (T2D) is caused by loss of pancreatic ß-cell mass and failure of the remaining ß-cells to deliver sufficient insulin to meet demand. ß-Cell glucolipotoxicity (GLT), which refers to combined, deleterious effects of elevated glucose and fatty acid levels on ß-cell function and survival, contributes to T2D-associated ß-cell failure. Drugs and mechanisms that protect ß-cells from GLT stress could potentially improve metabolic control in patients with T2D. In a phenotypic screen seeking low-molecular-weight compounds that protected ß-cells from GLT, we identified compound A that selectively blocked GLT-induced apoptosis in rat insulinoma cells. Compound A and its optimized analogs also improved viability and function in primary rat and human islets under GLT. We discovered that compound A analogs decreased GLT-induced cytosolic calcium influx in islet cells, and all measured ß-cell-protective effects correlated with this activity. Further studies revealed that the active compound from this series largely reversed GLT-induced global transcriptional changes. Our results suggest that taming cytosolic calcium overload in pancreatic islets can improve ß-cell survival and function under GLT stress and thus could be an effective strategy for T2D treatment.

Structure of lipoprotein lipase in complex with GPIHBP1.

Lipoprotein lipase (LPL) plays a central role in triglyceride (TG) metabolism. By catalyzing the hydrolysis of TGs present in TG-rich lipoproteins (TRLs), LPL facilitates TG utilization and regulates circulating TG and TRL concentrations. Until very recently, structural information for LPL was limited to homology models, presumably due to the propensity of LPL to unfold and aggregate. By coexpressing LPL with a soluble variant of its accessory protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) and with its chaperone protein lipase maturation factor 1 (LMF1), we obtained a stable and homogenous LPL/GPIHBP1 complex that was suitable for structure determination. We report here X-ray crystal structures of human LPL in complex with human GPIHBP1 at 2.5-3.0 Å resolution, including a structure with a novel inhibitor bound to LPL. Binding of the inhibitor resulted in ordering of the LPL lid and lipid-binding regions and thus enabled determination of the first crystal structure of LPL that includes these important regions of the protein. It was assumed for many years that LPL was only active as a homodimer. The structures and additional biochemical data reported here are consistent with a new report that LPL, in complex with GPIHBP1, can be active as a monomeric 1:1 complex. The crystal structures illuminate the structural basis for LPL-mediated TRL lipolysis as well as LPL stabilization and transport by GPIHBP1.

Small-molecule factor B inhibitor for the treatment of complement-mediated diseases.

Dysregulation of the alternative complement pathway (AP) predisposes individuals to a number of diseases including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Moreover, glomerular Ig deposits can lead to complement-driven nephropathies. Here we describe the discovery of a highly potent, reversible, and selective small-molecule inhibitor of factor B, a serine protease that drives the central amplification loop of the AP. Oral administration of the inhibitor prevents KRN-induced arthritis in mice and is effective upon prophylactic and therapeutic dosing in an experimental model of membranous nephropathy in rats. In addition, inhibition of factor B prevents complement activation in sera from C3 glomerulopathy patients and the hemolysis of human PNH erythrocytes. These data demonstrate the potential therapeutic value of using a factor B inhibitor for systemic treatment of complement-mediated diseases and provide a basis for its clinical development.

Fragment-Based Drug Discovery of Inhibitors of Phosphopantetheine Adenylyltransferase from Gram-Negative Bacteria.

The discovery and development of new antibiotics capable of curing infections due to multidrug-resistant and pandrug-resistant Gram-negative bacteria are a major challenge with fundamental importance to our global healthcare system. Part of our broad program at Novartis to address this urgent, unmet need includes the search for new agents that inhibit novel bacterial targets. Here we report the discovery and hit-to-lead optimization of new inhibitors of phosphopantetheine adenylyltransferase (PPAT) from Gram-negative bacteria. Utilizing a fragment-based screening approach, we discovered a number of unique scaffolds capable of interacting with the pantetheine site of E. coli PPAT and inhibiting enzymatic activity, including triazolopyrimidinone 6. Structure-based optimization resulted in the identification of two lead compounds as selective, small molecule inhibitors of bacterial PPAT: triazolopyrimidinone 53 and azabenzimidazole 54 efficiently inhibited E. coli and P. aeruginosa PPAT and displayed modest cellular potency against the efflux-deficient E. coli Δ tolC mutant strain.

Syringe Filling of High-Concentration mAb Formulation: Slow Suck-Back Pump Speed Prevented Filling Needle Clogging.

Partial and complete clogging of filling needles occurred during syringe filling of a high-concentration mAb formulation. This caused nonvertical liquid flow, which eventually led to the termination of filling. Overcoming this phenomenon was essential to ensure minimal fill weight variation, product waste, and manufacturing downtime. The liquid behavior inside the filling needle was studied using glass and stainless steel needles and demonstrated that effective suck-back control was critical for preventing needle clogging. A key finding of our work is that the suck-back pump speed was a critical factor to achieve an effective suck back. More specifically, a slow suck-back pump speed (<10 rpm; liquid flow rate, <5 mL/min) was essential to improve suck-back control inside the conventional stainless steel filling needles. In contrast, higher suck-back pump speeds (>10 rpm; liquid flow rate, >5 mL/min) resulted in downward product migration within the filling needle leading to formation of a liquid plug at the needle tip, which was prone to rapid drying. Slowing the suck-back pump speed in conjunction with modifying the suck-back volume was effective at consistently withdrawing product into the stainless steel filling needles and prevented needle clogging.

A Fluorescence-Based High-Throughput Screening Assay to Identify Growth Inhibitors of the Pathogenic Fungus Aspergillus fumigatus.

Due to the advancements in modern medicine that have resulted in an increased number of immunocompromised individuals, the incidences and the associated mortality of invasive aspergillosis have continued to rise over the past three decades despite appropriate treatment. As a result, invasive aspergillosis has emerged as a leading cause of infection-related mortality in immunocompromised individuals. Utilizing the resazurin to resorufin conversion fluorescence readout to monitor cell viability, herein, we outline a high-throughput screening method amenable to profiling a large pharmaceutical library against the clinically relevant but less frequently screened fungal pathogen Aspergillus fumigatus. This enables the user to conduct high-throughput screening using a disease-relevant fungal growth assay and identify novel antifungal chemotypes as drug leads.

SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice.

Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene, represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5' splice site is increased in a sequence-selective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule-mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases.

Identifying initiation and elongation inhibitors of dengue virus RNA polymerase in a high-throughput lead-finding campaign.

Dengue virus (DENV) is the most significant mosquito-borne viral pathogen in the world and is the cause of dengue fever. The DENV RNA-dependent RNA polymerase (RdRp) is conserved among the four viral serotypes and is an attractive target for antiviral drug development. During initiation of viral RNA synthesis, the polymerase switches from a "closed" to "open" conformation to accommodate the viral RNA template. Inhibitors that lock the "closed" or block the "open" conformation would prevent viral RNA synthesis. Herein, we describe a screening campaign that employed two biochemical assays to identify inhibitors of RdRp initiation and elongation. Using a DENV subgenomic RNA template that promotes RdRp de novo initiation, the first assay measures cytosine nucleotide analogue (Atto-CTP) incorporation. Liberated Atto fluorophore allows for quantification of RdRp activity via fluorescence. The second assay uses the same RNA template but is label free and directly detects RdRp-mediated liberation of pyrophosphates of native ribonucleotides via liquid chromatography-mass spectrometry. The ability of inhibitors to bind and stabilize a "closed" conformation of the DENV RdRp was further assessed in a differential scanning fluorimetry assay. Last, active compounds were evaluated in a renilla luciferase-based DENV replicon cell-based assay to monitor cellular efficacy. All assays described herein are medium to high throughput, are robust and reproducible, and allow identification of inhibitors of the open and closed forms of DENV RNA polymerase.

Timing of neuroprognostication in postcardiac arrest therapeutic hypothermia*.

OBJECTIVE: Early assessment of neurologic recovery is often challenging in survivors of cardiac arrest. Further, little is known about when to assess neurologic status in comatose, postarrest patients receiving therapeutic hypothermia. We sought to evaluate timing of prognostication in cardiac arrest survivors who received therapeutic hypothermia. DESIGN: A retrospective chart review of consecutive postarrest patients receiving therapeutic hypothermia (protocol: 24-hr maintenance at target temperature followed by rewarming over 8 hrs). Data were abstracted from the medical chart, including documentation during the first 96 hrs post arrest of "poor" prognosis, diagnostic tests for neuroprognostication, consultations used for determination of prognosis, and outcome at discharge. SETTING: Two academic urban emergency departments. PATIENTS: A total of 55 consecutive patients who underwent therapeutic hypothermia were reviewed between September 2005 and April 2009. INTERVENTION: None. RESULTS: Of our cohort of comatose postarrest patients, 59% (29 of 49) were male, and the mean age was 56 ± 16 yrs. Chart documentation of "poor" or "grave" prognosis occurred "early": during induction, maintenance of cooling, rewarming, or within 15 hrs after normothermia in 57% (28 of 49) of cases. Of patients with early documentation of poor prognosis, 25% (seven of 28) had care withdrawn within 72 hrs post arrest, and 21% (six of 28) survived to discharge with favorable neurologic recovery. In the first 96 hrs post arrest: 88% (43 of 49) of patients received a head computed tomography, 90% (44 of 49) received electroencephalography, 2% (one of 49) received somatosensory evoked potential testing, and 71% (35 of 49) received neurology consultation. CONCLUSIONS: Documentation of "poor prognosis" occurred during therapeutic hypothermia in more than half of patients in our cohort. Premature documentation of poor prognosis may contribute to early decisions to withdraw care. Future guidelines should address when to best prognosticate in postarrest patients receiving therapeutic hypothermia.

The validity of using administrative claims data in total joint arthroplasty outcomes research.

The purpose of this study was to evaluate concordance between administrative and clinical diagnosis and procedure codes for revision total joint arthroplasty (TJA). Concordance between administrative and clinical records was determined for 764 consecutive revision TJA procedures from 4 hospitals. For revision total hip arthroplasty, concordance between clinical diagnoses and administrative claims was very good for dislocation, mechanical loosening, and periprosthetic joint infection (all kappa > 0.6), but considerably lower for prosthetic implant failure/breakage and other mechanical complication (both kappa < 0.25). Similarly, for revision total knee arthroplasty diagnoses, concordance was very good for periprosthetic fracture, periprosthetic joint infection, mechanical loosening, and osteolysis (all kappa > 0.60), but much lower for implant failure/breakage and other mechanical complication (both kappa < 0.24). Concordance for TJA-specific procedure codes was very good only for revision total knee arthroplasty patellar component revisions and tibial insert exchange procedures. Total (all-component) revisions were overcoded for hips (00.70) and undercoded for knees (00.80). Improved clinical documentation and continued education are needed to enhance the value of these codes.

Cell phone cardiopulmonary resuscitation: audio instructions when needed by lay rescuers: a randomized, controlled trial.

STUDY OBJECTIVE: Given the ubiquitous presence of cellular telephones, we seek to evaluate the extent to which prerecorded audio cardiopulmonary resuscitation (CPR) instructions delivered by a cell telephone will improve the quality of CPR provided by untrained and trained lay rescuers. METHODS: We randomly assigned both previously CPR trained and untrained volunteers to perform CPR on a manikin for 3 minutes with or without audio assistance from a cell telephone programmed to provide CPR instructions. We measured CPR quality metrics-pauses (ie, no flow time), compression rate (minute), depth (millimeters), and hand placement (percentage correct)-across the 4 groups defined by being either CPR trained or untrained and receiving or not receiving cell telephone CPR instructions. RESULTS: There was no difference in CPR measures for participants who had or had not received previous CPR training. Participants using the cell telephone aid performed better compression rate (100/minute [95% confidence interval (CI) 97 to 103/minute] versus 44/minute [95% CI 38 to 50/minute]), compression depth (41 mm [95% CI 38 to 44 mm] versus 31 mm [95% CI 28 to 34 mm]), hand placement (97% [95% CI 94% to 100%] versus 75% [95% CI 68% to 83%] correct), and fewer pauses (74 seconds [95% CI 72 to 76 seconds] versus 89 seconds [95% CI 80 to 98 seconds]) compared with participants without the cell telephone aid. CONCLUSION: A simple audio program that can be made available for cell telephones increases the quality of bystander CPR in a manikin simulation.

A rodent model of emergency cardiopulmonary bypass resuscitation with different temperatures after asphyxial cardiac arrest.

BACKGROUND: The use of emergency cardiopulmonary bypass (ECPB) resuscitation after cardiac arrest may offer hope for survival when standard ACLS therapies fail. However, whether cooling adds benefit to ECPB is unknown and we lack an ECPB rodent model for experimental studies. We sought to (a) develop a 72 h survival rodent model using ECPB to treat asphyxial cardiac arrest and (b) use this new model to evaluate early mild and moderate hypothermia versus normothermia during ECPB resuscitation. METHODS: After 8 min of normothermic asphyxia, three groups of rats were resuscitated with ECPB at 37 degrees C (NORM), 34 degrees C (MILD) and 30 degrees C (MOD) for 1h (n=10 each). During the second resuscitation hour, ECPB was discontinued, ventilatory support was provided and body temperatures were maintained at 37 degrees C for NORM, 34 degrees C for MILD, and from 30 degrees C gradually up to 34 degrees C in 1h for MOD animals. From hours 3 to 8, body temperature was maintained at 37 degrees C for NORM and 34 degrees C for MILD and MOD animals. RESULTS: All rats were initially resuscitated by ECPB. After 72 h, neurological outcome and survival in the MILD (60% survival) and MOD (80%) groups were significantly better than in the NORM (0%) group (p<0.05). Overall performance recovery in the MOD group was best (vs. the NORM group), while the MILD group had an intermediate outcome. CONCLUSIONS: A rodent model of ECPB is feasible and useful for resuscitation studies. The addition of early mild and moderate hypothermia to ECPB resuscitation significantly improves survival compared with normothermic ECPB in rats.

Identification of peptide antagonists to glycoprotein Ibalpha that selectively inhibit von Willebrand factor dependent platelet aggregation.

GPIbalpha is an integral membrane protein of the GPIb-IX-V complex found on the platelet surface that interacts with the A1 domain of von Willebrand factor (vWF-A1). The interaction of GPIbalpha with vWF-A1 under conditions of high shear stress is the first step in platelet-driven thrombus formation. Phage display was used to identify peptide antagonists of the GPIbalpha-vWF-A1 interaction. Two nine amino acid cysteine-constrained phage display libraries were screened against GPIbalpha revealing peptides that formed a consensus sequence. A peptide with sequence most representative of the consensus, designated PS-4, was used as the basis for an optimized library. The optimized selection identified additional GPIbalpha binding peptides with sequences nearly identical to the parent peptide. Surface plasmon resonance of the PS-4 parent and two optimized synthetic peptides, OS-1 and OS-2, determined their equilibrium dissociation GPIbalpha binding constants ( K Ds) of 64, 0.74, and 31 nM, respectively. Isothermal calorimetry corroborated the K D of peptide PS-4 with a resulting affinity value of 68 nM. An ELISA demonstrated that peptides PS-4, OS-1, and OS-2 competitively inhibited the interaction between the vWF-A1 domain and GPIbalpha-Fc in a concentration-dependent manner. All three peptides inhibited GPIbalpha-vWF-mediated platelet aggregation induced under high shear conditions using the platelet function analyzer (PFA-100) with full blockade observed at 150 nM for OS-1. In addition, OS-1 blocked ristocetin-induced platelet agglutination of human platelets in plasma with no influence on platelet aggregation induced by several agonists of alternative platelet aggregation pathways, demonstrating that this peptide specifically disrupted the GPIbalpha-vWF-A1 interaction.