Clinical targeting of HIV capsid protein with a long-acting small molecule.

Oral antiretroviral agents provide life-saving treatments for millions of people living with HIV, and can prevent new infections via pre-exposure prophylaxis1-5. However, some people living with HIV who are heavily treatment-experienced have limited or no treatment options, owing to multidrug resistance6. In addition, suboptimal adherence to oral daily regimens can negatively affect the outcome of treatment-which contributes to virologic failure, resistance generation and viral transmission-as well as of pre-exposure prophylaxis, leading to new infections1,2,4,7-9. Long-acting agents from new antiretroviral classes can provide much-needed treatment options for people living with HIV who are heavily treatment-experienced, and additionally can improve adherence10. Here we describe GS-6207, a small molecule that disrupts the functions of HIV capsid protein and is amenable to long-acting therapy owing to its high potency, low in vivo systemic clearance and slow release kinetics from the subcutaneous injection site. Drawing on X-ray crystallographic information, we designed GS-6207 to bind tightly at a conserved interface between capsid protein monomers, where it interferes with capsid-protein-mediated interactions between proteins that are essential for multiple phases of the viral replication cycle. GS-6207 exhibits antiviral activity at picomolar concentrations against all subtypes of HIV-1 that we tested, and shows high synergy and no cross-resistance with approved antiretroviral drugs. In phase-1 clinical studies, monotherapy with a single subcutaneous dose of GS-6207 (450 mg) resulted in a mean log10-transformed reduction of plasma viral load of 2.2 after 9 days, and showed sustained plasma exposure at antivirally active concentrations for more than 6 months. These results provide clinical validation for therapies that target the functions of HIV capsid protein, and demonstrate the potential of GS-6207 as a long-acting agent to treat or prevent infection with HIV.

Discovery of velpatasvir (GS-5816): A potent pan-genotypic HCV NS5A inhibitor in the single-tablet regimens Vosevi® and Epclusa®.

Direct-acting antiviral inhibitors have revolutionized the treatment of hepatitis C virus (HCV) infected patients. Herein is described the discovery of velpatasvir (VEL, GS-5816), a potent pan-genotypic HCV NS5A inhibitor that is a component of the only approved pan-genotypic single-tablet regimens (STRs) for the cure of HCV infection. VEL combined with sofosbuvir (SOF) is Epclusa®, an STR with 98% cure-rates for genotype 1-6 HCV infected patients. Addition of the pan-genotypic HCV NS3/4A protease inhibitor voxilaprevir to SOF/VEL is the STR Vosevi®, which affords 97% cure-rates for genotype 1-6 HCV patients who have previously failed another treatment regimen.

Discovery of the pan-genotypic hepatitis C virus NS3/4A protease inhibitor voxilaprevir (GS-9857): A component of Vosevi®.

Treatment of hepatitis C virus (HCV) infection has been historically challenging due the high viral genetic complexity wherein there are eight distinct genotypes and at least 86 viral subtypes. While HCV NS3/4A protease inhibitors are an established treatment option for genotype 1 infection, limited coverage of genotypes 2 and/or 3 combined with serum alanine transaminase (ALT) elevations for some compounds has limited the broad utility of this therapeutic class. Our discovery efforts were focused on identifying an NS3/4A protease inhibitor with pan-genotypic antiviral activity, improved coverage of resistance associated substitutions, and a decreased risk of hepatotoxicity. Towards this goal, distinct interactions with the conserved catalytic triad of the NS3/4A protease were identified that improved genotype 3 antiviral activity. We further discovered that protein adduct formation strongly correlated with clinical ALT elevation for this therapeutic class. Improving metabolic stability and decreasing protein adduct formation through structural modifications ultimately resulted in voxilaprevir. Voxilaprevir, in combination with sofosbuvir and velpatasvir, has demonstrated pan-genotypic antiviral clinical activity. Furthermore, hepatotoxicity was not observed in Phase 3 clinical trials with voxilaprevir, consistent with our design strategy. Vosevi® (sofosbuvir, velpatasvir, and voxilaprevir) is now an approved pan-genotypic treatment option for the most difficult-to-cure individuals who have previously failed direct acting antiviral therapy.

Neratinib after trastuzumab-based adjuvant therapy in HER2-positive breast cancer (ExteNET): 5-year analysis of a randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: ExteNET showed that 1 year of neratinib, an irreversible pan-HER tyrosine kinase inhibitor, significantly improves 2-year invasive disease-free survival after trastuzumab-based adjuvant therapy in women with HER2-positive breast cancer. We report updated efficacy outcomes from a protocol-defined 5-year follow-up sensitivity analysis and long-term toxicity findings. METHODS: In this ongoing randomised, double-blind, placebo-controlled, phase 3 trial, eligible women aged 18 years or older (≥20 years in Japan) with stage 1-3c (modified to stage 2-3c in February, 2010) operable breast cancer, who had completed neoadjuvant and adjuvant chemotherapy plus trastuzumab with no evidence of disease recurrence or metastatic disease at study entry. Patients who were eligible patients were randomly assigned (1:1) via permuted blocks stratified according to hormone receptor status (hormone receptor-positive vs hormone receptor-negative), nodal status (0 vs 1-3 vs or ≥4 positive nodes), and trastuzumab adjuvant regimen (given sequentially vs concurrently with chemotherapy), then implemented centrally via an interactive voice and web-response system, to receive 1 year of oral neratinib 240 mg/day or matching placebo. Treatment was given continuously for 1 year, unless disease recurrence or new breast cancer, intolerable adverse events, or consent withdrawal occurred. Patients, investigators, and trial funder were masked to treatment allocation. The predefined endpoint of the 5-year analysis was invasive disease-free survival, analysed by intention to treat. ExteNET is registered with ClinicalTrials.gov, number NCT00878709, and is closed to new participants. FINDINGS: Between July 9, 2009, and Oct 24, 2011, 2840 eligible women with early HER2-positive breast cancer were recruited from community-based and academic institutions in 40 countries and randomly assigned to receive neratinib (n=1420) or placebo (n=1420). After a median follow-up of 5·2 years (IQR 2·1-5·3), patients in the neratinib group had significantly fewer invasive disease-free survival events than those in the placebo group (116 vs 163 events; stratified hazard ratio 0·73, 95% CI 0·57-0·92, p=0·0083). The 5-year invasive disease-free survival was 90·2% (95% CI 88·3-91·8) in the neratinib group and 87·7% (85·7-89·4) in the placebo group. Without diarrhoea prophylaxis, the most common grade 3-4 adverse events in the neratinib group, compared with the placebo group, were diarrhoea (561 [40%] grade 3 and one [<1%] grade 4 with neratinib vs 23 [2%] grade 3 with placebo), vomiting (grade 3: 47 [3%] vs five [<1%]), and nausea (grade 3: 26 [2%] vs two [<1%]). Treatment-emergent serious adverse events occurred in 103 (7%) women in the neratinib group and 85 (6%) women in the placebo group. No evidence of increased risk of long-term toxicity or long-term adverse consequences of neratinib-associated diarrhoea were identified with neratinib compared with placebo. INTERPRETATION: At the 5-year follow-up, 1 year of extended adjuvant therapy with neratinib, administered after chemotherapy and trastuzumab, significantly reduced the proportion of clinically relevant breast cancer relapses-ie, those that might lead to death, such as distant and locoregional relapses outside the preserved breast-without increasing the risk of long-term toxicity. An analysis of overall survival is planned after 248 events. FUNDING: Wyeth, Pfizer, and Puma Biotechnology.

Preclinical Pharmacokinetics and First-in-Human Pharmacokinetics, Safety, and Tolerability of Velpatasvir, a Pangenotypic Hepatitis C Virus NS5A Inhibitor, in Healthy Subjects.

Preclinical characterization of velpatasvir (VEL; GS-5816), an inhibitor of the hepatitis C virus (HCV) NS5A protein, demonstrated that it has favorable in vitro and in vivo properties, including potent antiviral activity against hepatitis C virus genotype 1 to 6 replicons, good metabolic stability, low systemic clearance, and adequate bioavailability and physicochemical properties, to warrant clinical evaluation. The phase 1 (first-in-human) study evaluated the safety, tolerability, and pharmacokinetics of VEL in healthy human subjects following administration of single and multiple (n = 7) once-daily ascending doses and of VEL in the presence and absence of food. Following administration of single and multiple doses, VEL was safe and well tolerated when administered at up to 450 mg and when administered with food. The pharmacokinetic behavior of VEL observed in humans was generally in agreement with that seen during preclinical characterization. Following administration of multiple doses, VEL trough concentrations were significantly greater than the protein-adjusted half-maximal (50%) effective concentration of VEL against HCV genotype 1 to 6 replicons at all evaluated doses greater than 5 mg. The pharmacokinetics of VEL were not significantly affected by administration with food. Collectively, the results of this study support the further clinical investigation of VEL administered once daily as part of a regimen with other pangenotypic direct-acting antivirals for the treatment of HCV infection.

Clinical Resistance to Velpatasvir (GS-5816), a Novel Pan-Genotypic Inhibitor of the Hepatitis C Virus NS5A Protein.

Velpatasvir (VEL, GS-5816) is a novel pan-genotypic hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor with activity against genotype 1 (GT1) to GT6 HCV replicons. In a phase 1b 3-day monotherapy study, patients treated with a 150-mg dose of GS-5816 had a mean maximal HCV RNA decline of ≥3.3 log10 IU/ml in GT1a, -1b, -2, -3, and -4. This report characterizes virologic resistance to VEL in these patients. NS5A resistance-associated substitutions (RASs) were detected by deep sequencing (1% cutoff) pretreatment in 22/70 patients, i.e., 10/35 (29%) patients with GT1a, 1/8 (13%) with GT1b, 4/8 (50.0%) with GT2, 5/17 (29.4%) with GT3, and 2/2 (100.0%) with GT4. In GT1a and GT3 patients, pretreatment RASs were associated with a slightly reduced HCV RNA response compared to that of patients without pretreatment RASs; among patients with GT1b, GT2, and GT4, no significant difference in response was observed in those with or without pretreatment RASs. Following treatment, the pattern of emergent RASs was more complex for GT1a than for the other genotypes. In GT1a, substitutions emerged at positions M28, Q30, L31, P32, H58, E92, and Y93, with the most prevalent substitutions at positions Y93, M28, and L31. RASs were observed at two positions in GT1b and GT2 (Y93 and L31), three positions in GT3 (Y93, L31, and E92), and four positions in GT4 (L28, M31, P32L, and Y93). RASs that were present pretreatment persisted through the 48-week follow-up period; however, RASs emerging during treatment were more likely to decline both in prevalence and in frequency within the viral population during follow-up. (This study has been registered at ClinicalTrials.gov under registration no. NCT01740791.).

In Vitro Antiviral Activity and Resistance Profile Characterization of the Hepatitis C Virus NS5A Inhibitor Ledipasvir.

Ledipasvir (LDV; GS-5885), a component of Harvoni (a fixed-dose combination of LDV with sofosbuvir [SOF]), is approved to treat chronic hepatitis C virus (HCV) infection. Here, we report key preclinical antiviral properties of LDV, including in vitro potency, in vitro resistance profile, and activity in combination with other anti-HCV agents. LDV has picomolar antiviral activity against genotype 1a and genotype 1b replicons with 50% effective concentration (EC50) values of 0.031 nM and 0.004 nM, respectively. LDV is also active against HCV genotypes 4a, 4d, 5a, and 6a with EC50 values of 0.11 to 1.1 nM. LDV has relatively less in vitro antiviral activity against genotypes 2a, 2b, 3a, and 6e, with EC50 values of 16 to 530 nM. In vitro resistance selection with LDV identified the single Y93H and Q30E resistance-associated variants (RAVs) in the NS5A gene; these RAVs were also observed in patients after a 3-day monotherapy treatment. In vitro antiviral combination studies indicate that LDV has additive to moderately synergistic antiviral activity when combined with other classes of HCV direct-acting antiviral (DAA) agents, including NS3/4A protease inhibitors and the nucleotide NS5B polymerase inhibitor SOF. Furthermore, LDV is active against known NS3 protease and NS5B polymerase inhibitor RAVs with EC50 values equivalent to those for the wild type.

Discovery of Hepatitis B Virus Surface Antigen Suppressor GS-8873.

Chronic hepatitis B (CHB) virus infection afflicts hundreds of millions of people and causes nearly one million deaths annually. The high levels of circulating viral surface antigen (HBsAg) that characterize CHB may lead to T-cell exhaustion, resulting in an impaired antiviral immune response in the host. Agents that suppress HBsAg could help invigorate immunity toward infected hepatocytes and facilitate a functional cure. A series of dihydropyridoisoquinolizinone (DHQ) inhibitors of human poly(A) polymerases PAPD5/7 were reported to suppress HBsAg in vitro. An example from this class, RG7834, briefly entered the clinic. We set out to identify a potent, orally bioavailable, and safe PAPD5/7 inhibitor as a potential component of a functional cure regimen. Our efforts led to the identification of a dihydropyridophthalazinone (DPP) core with improved pharmacokinetic properties. A conformational restriction strategy and optimization of core substitution led to GS-8873, which was projected to provide deep HBsAg suppression with once-daily dosing.

A phase 1, randomized, placebo-controlled, 3-day, dose-ranging study of GS-5885, an NS5A inhibitor, in patients with genotype 1 hepatitis C.

BACKGROUND & AIMS: GS-5885 is an inhibitor of the hepatitis C virus (HCV) NS5A protein and exhibits potent suppression of genotype 1 HCV replicons. The safety, tolerability, pharmacokinetics, antiviral activity, and resistance profile of once-daily GS-5885 doses of 1-90 mg were evaluated in patients with chronic genotype 1 HCV. METHODS: Genotype 1 HCV-infected patients were randomized to 3 days of once-daily (QD) dosing with placebo (n=12) or GS-5885 1 mg (n=10), 3 mg (n=10), 10 mg (n=20), 30 mg (n=10), or 90 mg (n=10). Plasma samples for pharmacokinetics, HCV RNA, and NS5A sequencing were collected through day 14. RESULTS: GS-5885 was well tolerated and resulted in median maximal reductions in HCV RNA ranging from 2.3 log(10) IU/ml (1 mg QD) to 3.3 log(10) IU/ml (10 mg QD in genotype 1b and 30 mg QD). E(max) modeling indicated GS-5885 30 mg was associated with>95% of maximal antiviral response to HCV genotype 1a. HCV RNA reductions were generally more sustained among patients with genotype 1b vs. 1a. Three of 60 patients had a reduced response and harbored NS5A-resistant virus at baseline. NS5A sequencing identified residues 30 and 31 in genotype 1a, and 93 in genotype 1b as the predominant sites of mutation following GS-5885 dosing. Plasma pharmacokinetics was consistent with QD dosing. CONCLUSIONS: During 3 days of monotherapy, low doses of GS-5885 demonstrated significant antiviral activity in genotype 1a and 1b HCV-infected patients. GS-5885 is currently being evaluated in combination with direct antiviral regimens with and without peginterferon.

Discovery of GS-9451: an acid inhibitor of the hepatitis C virus NS3/4A protease.

The discovery of GS-9451 is reported. Modification of the P3 cap and P2 quinoline with a series of solubilizing groups led to the identification of potent HCV NS3 protease inhibitors with greatly improved pharmacokinetic properties in rats, dogs and monkeys.

Final Efficacy Results of Neratinib in HER2-positive Hormone Receptor-positive Early-stage Breast Cancer From the Phase III ExteNET Trial.

BACKGROUND: The ExteNET trial demonstrated improved invasive disease-free survival (iDFS) with neratinib, an irreversible pan-HER tyrosine kinase inhibitor, versus placebo in patients with human epidermal growth factor receptor 2-positive (HER2+)/hormone receptor-positive (HR+) early-stage breast cancer (eBC). PATIENTS AND METHODS: ExteNET was a multicenter, randomized, double-blind, phase III trial of 2840 patients with HER2+ eBC after neoadjuvant/adjuvant trastuzumab-based therapy. Patients were stratified by HR status and randomly assigned 1-year oral neratinib 240 mg/day or placebo. The primary endpoint was iDFS. Descriptive analyses were performed in patients with HR+ eBC who initiated treatment ≤ 1 year (HR+/≤ 1-year) and > 1 year (HR+/> 1-year) post-trastuzumab. RESULTS: HR+/≤ 1-year and HR+/> 1-year populations comprised 1334 (neratinib, n = 670; placebo, n = 664) and 297 (neratinib, n = 146; placebo, n = 151) patients, respectively. Absolute iDFS benefits at 5 years were 5.1% in HR+/≤ 1-year (hazard ratio, 0.58; 95% confidence interval [CI], 0.41-0.82) and 1.3% in HR+/>1-year (hazard ratio, 0.74; 95% CI, 0.29-1.84). In HR+/≤ 1-year, neratinib was associated with a numerical improvement in overall survival (OS) at 8 years (absolute benefit, 2.1%; hazard ratio, 0.79; 95% CI, 0.55-1.13). Of 354 patients in the HR+/≤ 1-year group who received neoadjuvant therapy, 295 had residual disease, and results showed absolute benefits of 7.4% at 5-year iDFS (hazard ratio, 0.60; 95% CI, 0.33-1.07) and 9.1% at 8-year OS (hazard ratio, 0.47; 95% CI, 0.23-0.92). There were fewer central nervous system events with neratinib. Adverse events were similar to those previously reported. CONCLUSION: Neratinib significantly improved iDFS in the HER2+/HR+/≤ 1-year population, and a similar trend was observed in patients with residual disease following neoadjuvant treatment. Numerical improvements in central nervous system events and OS were consistent with iDFS benefits and suggest long-term benefit for neratinib in this population.

Lessons learned from 14 years of outcomes: the need for collaboration, utilization, and projection.

In 1995, the Indiana Association of Residential Child Care Agencies (IARCCA), an association of children and family services, responded to a request to demonstrate effectiveness of residential care. The organization developed a vision for evaluating outcomes that incorporated collaboration with others, use of data and projection toward the future. This guiding vision led to an ongoing project for IARCCA member agencies across Indiana. This article shares lessons learned from this 15-year process, including developing benchmarks to establish best practices for serving youth.

Level of HER-2/neu protein expression in breast cancer may affect the development of endogenous HER-2/neu-specific immunity.

We questioned whether the incidence or magnitude of the humoral or cellular immune response to the self-tumor antigen HER-2/neu is influenced by the level of HER-2/neu protein overexpression as defined by immunohistochemical staining of tumors in breast cancer patients. We obtained peripheral blood from 104 women with stage II, III, and IV pathologically confirmed HER-2/neu-overexpressing breast cancer. Patients were categorized with +1 (n = 14), +2 (n = 20), or +3 (n = 70) HER-2/neu overexpression by institutional pathologic report. Circulating antibodies to HER-2/neu were evaluated using ELISA. T-cell responses to HER-2/neu were measured using an antigen-specific tritiated thymidine incorporation assay. Eighty-two percent of subjects with HER-2/neu antibodies were +3 overexpressors compared with 18% +2 overexpressors and 0% +1 overexpressors, a highly significant difference (P < 0.001), and there were significant differences in the magnitude of the HER-2/neu-specific antibodies between groups with varying HER-2/neu protein expression (P = 0.022). In addition, 65% of subjects with HER-2/neu-specific T cells were +3 overexpressors compared with 16% +2 overexpressors and 19% +1 overexpressors (P = 0.001). Data presented here indicate that endogenous HER-2/neu-specific humoral and T-cell immunity is greater in patients with +3 protein overexpression in their tumors than in patients with lower levels of HER-2/neu expression. Overexpression of a self-tumor-associated protein is a potential mechanism by which immunogenicity is enhanced and may aid in the identification of biologically relevant proteins to target for immune-based molecular cancer therapies.

A highly potent long-acting small-molecule HIV-1 capsid inhibitor with efficacy in a humanized mouse model.

People living with HIV (PLWH) have expressed concern about the life-long burden and stigma associated with taking pills daily and can experience medication fatigue that might lead to suboptimal treatment adherence and the emergence of drug-resistant viral variants, thereby limiting future treatment options1-3. As such, there is strong interest in long-acting antiretroviral (ARV) agents that can be administered less frequently4. Herein, we report GS-CA1, a new archetypal small-molecule HIV capsid inhibitor with exceptional potency against HIV-2 and all major HIV-1 types, including viral variants resistant to the ARVs currently in clinical use. Mechanism-of-action studies indicate that GS-CA1 binds directly to the HIV-1 capsid and interferes with capsid-mediated nuclear import of viral DNA, HIV particle production and ordered capsid assembly. GS-CA1 selects in vitro for unfit GS-CA1-resistant capsid variants that remain fully susceptible to other classes of ARVs. Its high metabolic stability and low solubility enabled sustained drug release in mice following a single subcutaneous dosing. GS-CA1 showed high antiviral efficacy as a long-acting injectable monotherapy in a humanized mouse model of HIV-1 infection, outperforming long-acting rilpivirine. Collectively, these results demonstrate the potential of ultrapotent capsid inhibitors as new long-acting agents for the treatment of HIV-1 infection.

Carboplatin plus gemcitabine repeating doublet therapy in recurrent breast cancer.

PURPOSE: The combination of cisplatin plus gemcitabine is active in metastatic breast cancer. Carboplatin plus gemcitabine, widely used in ovarian and non-small-cell lung cancers, has also been used in breast cancer. This trial examined the efficacy and toxicity of split-dose carboplatin plus gemcitabine in advanced breast cancer. PATIENTS AND METHODS: Patients with measurable disease, recurrent after adjuvant and < or = 1 previous treatment for systemic disease, received carboplatin area under the curve = 2.0 (Calvert) plus gemcitabine 800 mg/m2, both drugs administered days 1 and 8 every 21 days. Of 15 patients accrued, 13 are fully evaluable. RESULTS: There were 2 complete (13.3%) and 6 partial (40%) responses, for an overall response rate by intention to treat of 53.3% (95% CI, 28%-82%). The median time to progression was 4.5 months (95% CI, 2.03-6.97 months), and median overall survival was 28.8 months (95% CI, 9.4-48.2 months). There were 2 patients with grade 3 (13.3%) anemia, 7 patients with grade 3 (46.6%) and 4 patients (26.6%) with grade 4 neutropenia, 4 patients with grade 3 (26.6%) and 3 patients (20%) with grade 4 thrombocytopenia. CONCLUSION: The repeating doublet of split-dose carboplatin plus gemcitabine reveals activity comparable to that of cisplatin plus gemcitabine, is well tolerated, and warrants evaluation in patients with recurrent breast cancer.

Bevacizumab and albumin-bound paclitaxel treatment in metastatic breast cancer.

BACKGROUND: Miller et al demonstrated that the combination of bevacizumab and paclitaxel has significant activity in metastatic breast cancer (MBC). Because albumin-bound paclitaxel has been shown to have less toxicity, a better tumor delivery, and possibly better response for MBC, we combined it with bevacizumab to treat women with MBC. PATIENTS AND METHODS: This is a retrospective analysis. Billing records from March 2005 through December 2006 were reviewed to identify all patients treated with a combination of albumin-bound paclitaxel/bevacizumab. A total of 40 women were identified. They received a minimum of 2 courses. Patients with measurable disease were monitored for response using Response Evaluation Criteria in Solid Tumors. Women with bone-only disease were monitored with positron emission tomography (PET)/computed tomography/magnetic resonance imaging and tumor markers. All response data were confirmed by independent review. RESULTS: Of 33 women with measurable disease, 16 had objective responses to the albumin-bound paclitaxel/bevacizumab regimen (3 complete responses and 13 partial responses) for an overall response rate (ORR) of 48.5%. Median time to progression for responders was 128 days. Another 5 women had stable disease (SD) with a median duration of 135 days. Of 7 patients with bone-only disease, 2 had almost complete resolution of PET activity and 4 had SD (median, 148 days). Toxicity was acceptable with fatigue, neuropathy, pain, and hypertension being the most common complaints. CONCLUSION: In our limited series of women with advanced, heavily pretreated MBC treated with albumin-bound paclitaxel/bevacizumab, we saw a 48.5% ORR. The regimen was well tolerated. Randomized studies are needed to confirm efficacy and safety of this combination in treating breast cancer.

Gene expression changes by amyloid beta peptide-stimulated human postmortem brain microglia identify activation of multiple inflammatory processes.

A central feature of the inflammatory pathology in Alzheimer's disease is activated microglia clustered around aggregated amyloid beta (Abeta) peptide-containing plaques. In vitro-cultured microglia can be activated to an inflammatory state by aggregated Abeta with the induction of a range of different neurotoxic factors and provide a model system for studying microglia Abeta interactions. Gene expression responses of human postmortem brain-derived microglia to aggregated Abeta were measured using whole genome microarrays to address the hypothesis that Abeta interactions with human microglia primarily induce proinflammatory genes and not activation of genes involved in Abeta phagocytosis and removal. The results demonstrated that Abeta activation of microglia induced a large alteration in gene transcription including activation of many proinflammatory cytokines and chemokines, most notably, interleukin (IL)-1beta, IL-8, and matrix metalloproteinases (MMP), including MMP1, MMP3, MMP9, MMP10, and MMP12. All of these genes could amplify ongoing inflammation, resulting in further neuronal loss. Changes in expression of receptors associated with Abeta phagocytosis did not match the changes in proinflammatory gene expression. Time-course gene expression profiling, along with real-time polymerase chain reaction validation of expression changes, demonstrated an acute phase of gene induction for many proinflammatory genes but also chronic activation for many other potentially toxic products. These chronically activated genes included indoleamine 2,3-dioxygenase and kynureninase, which are involved in formation of the neurotoxin quinolinic acid, and S100A8, a potential proinflammatory chemokine. These studies show that activation of microglia by Abeta induces multiple genes that could be involved in inflammatory responses contributing to neurodegenerative processes.

Advances in cathepsin S inhibitor design.

Cathepsin S is expressed in antigen-presenting cells and plays a role in invariant chain processing and major histocompatibility complex class II (MHCII) antigen presentation leading to CD4+ T-cell activation. An oral cathepsin S inhibitor that blocks MHCII antigen presentation could result in a T-cell-selective immunosuppressant agent with improved safety over the current standard of care for the treatment of rheumatoid arthritis, psoriasis, multiple sclerosis and other autoimmune-based inflammatory diseases. This review focuses on advances in cathepsin S inhibitor utility and design since January of 2004.

Evaluation of a Fully Automated Research Prototype for the Immediate Identification of Microorganisms from Positive Blood Cultures under Clinical Conditions.

UNLABELLED: A clinical laboratory evaluation of an intrinsic fluorescence spectroscopy (IFS)-based identification system paired to a BacT/Alert Virtuo microbial detection system (bioMérieux, Inc., Durham, NC) was performed to assess the potential for fully automated identification of positive blood cultures. The prototype IFS system incorporates a novel method combining a simple microbial purification procedure with rapid in situ identification via spectroscopy. Results were available within 15 min of a bottle signaling positive and required no manual intervention. Among cultures positive for organisms contained within the database and producing acceptable spectra, 75 of 88 (85.2%) and 79 of 88 (89.8%) were correctly identified to the species and genus level, respectively. These results are similar to the performance of existing rapid methods. IMPORTANCE: A fully automated research platform was developed to identify microbial growth from positive blood cultures in <15 min. Because of the automated format, results can be generated during all shifts, with or without staffing, which in turn could promote more timely administration of target antimicrobial therapy.

Direct binding of ledipasvir to HCV NS5A: mechanism of resistance to an HCV antiviral agent.

Ledipasvir, a direct acting antiviral agent (DAA) targeting the Hepatitis C Virus NS5A protein, exhibits picomolar activity in replicon cells. While its mechanism of action is unclear, mutations that confer resistance to ledipasvir in HCV replicon cells are located in NS5A, suggesting that NS5A is the direct target of ledipasvir. To date co-precipitation and cross-linking experiments in replicon or NS5A transfected cells have not conclusively shown a direct, specific interaction between NS5A and ledipasvir. Using recombinant, full length NS5A, we show that ledipasvir binds directly, with high affinity and specificity, to NS5A. Ledipasvir binding to recombinant NS5A is saturable with a dissociation constant in the low nanomolar range. A mutant form of NS5A (Y93H) that confers resistance to ledipasvir shows diminished binding to ledipasvir. The current study shows that ledipasvir inhibits NS5A through direct binding and that resistance to ledipasvir is the result of a reduction in binding affinity to NS5A mutants.