Quantifying sit-to-stand and stand-to-sit transitions in free-living environments using the activPAL thigh-worn activity monitor.

PURPOSE: Standing up, sitting down and walking require considerable effort and coordination, which are crucial indicators to rehabilitation (e.g. stroke), and in older populations may indicate the onset of frailty and physical and cognitive decline. Currently, there are few reports robustly quantifying sit-to-stand and stand-to-sit transitions in free-living environments. The aim of this study was to identify and quantify these transitions using the peak velocity of sit-to-stand and stand-to-sit transitions to determine if these velocities were different in a healthy cohort and a mobility-impaired population. METHODS: Free-living sit-to-stand and stand-to-sit acceleration data were recorded from 21 healthy volunteers and 34 stroke survivors using activPAL3™ monitors over a one-week period. Thigh inclination velocity was calculated from these accelerometer data. Maximum velocities were compared between populations. RESULTS: A total of 10,299 and 11,392 sit-to-stand and stand-to-sit transitions were recorded in healthy volunteers and stroke survivors, respectively. Healthy volunteers had significantly higher overall mean peak velocities for both transitions compared with stroke survivors [70.7°/s ± 52.2 versus 44.2°/s ± 28.0 for sit-to-stand, P < 0.001 and 74.7°/s ± 51.8 versus 46.0°/s ± 31.9 for stand-to-sit; P < 0.001]. Mean peak velocity of transition was associated with increased variation in peak velocity across both groups. CONCLUSION: There were significant differences in the mean peak velocity of sit-to-stand and stand-to-sit transitions between the groups. Variation in an individual's mean peak velocity may be associated with the ability to perform these transitions. This method could be used to evaluate the effectiveness of interventions following injury such as stroke, as well as monitor decline in functional ability.

AGI-134: a fully synthetic α-Gal glycolipid that converts tumors into in situ autologous vaccines, induces anti-tumor immunity and is synergistic with an anti-PD-1 antibody in mouse melanoma models.

BACKGROUND: Treatments that generate T cell-mediated immunity to a patient's unique neoantigens are the current holy grail of cancer immunotherapy. In particular, treatments that do not require cumbersome and individualized ex vivo processing or manufacturing processes are especially sought after. Here we report that AGI-134, a glycolipid-like small molecule, can be used for coating tumor cells with the xenoantigen Galα1-3Galß1-4GlcNAc (α-Gal) in situ leading to opsonization with pre-existing natural anti-α-Gal antibodies (in short anti-Gal), which triggers immune cascades resulting in T cell mediated anti-tumor immunity. METHODS: Various immunological effects of coating tumor cells with α-Gal via AGI-134 in vitro were measured by flow cytometry: (1) opsonization with anti-Gal and complement, (2) antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells, and (3) phagocytosis and antigen cross-presentation by antigen presenting cells (APCs). A viability kit was used to test AGI-134 mediated complement dependent cytotoxicity (CDC) in cancer cells. The anti-tumoral activity of AGI-134 alone or in combination with an anti-programmed death-1 (anti-PD-1) antibody was tested in melanoma models in anti-Gal expressing galactosyltransferase knockout (α1,3GT-/-) mice. CDC and phagocytosis data were analyzed by one-way ANOVA, ADCC results by paired t-test, distal tumor growth by Mantel-Cox test, C5a data by Mann-Whitney test, and single tumor regression by repeated measures analysis. RESULTS: In vitro, α-Gal labelling of tumor cells via AGI-134 incorporation into the cell membrane leads to anti-Gal binding and complement activation. Through the effects of complement and ADCC, tumor cells are lysed and tumor antigen uptake by APCs increased. Antigen associated with lysed cells is cross-presented by CD8α+ dendritic cells leading to activation of antigen-specific CD8+ T cells. In B16-F10 or JB/RH melanoma models in α1,3GT-/- mice, intratumoral AGI-134 administration leads to primary tumor regression and has a robust abscopal effect, i.e., it protects from the development of distal, uninjected lesions. Combinations of AGI-134 and anti-PD-1 antibody shows a synergistic benefit in protection from secondary tumor growth. CONCLUSIONS: We have identified AGI-134 as an immunotherapeutic drug candidate, which could be an excellent combination partner for anti-PD-1 therapy, by facilitating tumor antigen processing and increasing the repertoire of tumor-specific T cells prior to anti-PD-1 treatment.

The discovery of potent nonstructural protein 5A (NS5A) inhibitors with a unique resistance profile-Part†1.

Nonstructural protein 5A (NS5A) represents a novel target for the treatment of hepatitis C virus (HCV). Daclatasvir, recently reported by Bristol-Myers-Squibb, is a potent NS5A inhibitor currently under investigation in phase 3 clinical trials. While the performance of daclatasvir has been impressive, the emergence of resistance could prove problematic and as such, improved analogues are being sought. By varying the biphenyl-imidazole unit of daclatasvir, novel inhibitors of HCV NS5A were identified with an improved resistance profile against mutant strains of the virus while retaining the picomolar potency of daclatasvir. One compound in particular, methyl ((S)-1-((S)-2-(4-(4-(6-(2-((S)-1-((methoxycarbonyl)-L-valyl)pyrrolidin-2-yl)-1H-imidazol-5-yl)quinoxalin-2-yl)phenyl)-1H-imidazol-2-yl)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)carbamate (17), exhibited very promising activity and showed good absorption and a long predicted human pharmacokinetic half-life. This compound represents a promising lead that warrants further evaluation.

The discovery of potent nonstructural protein 5A (NS5A) inhibitors with a unique resistance profile-Part†2.

In ongoing studies towards novel hepatitis C virus (HCV) therapeutics, inhibitors of nonstructural protein 5A (NS5A) were evaluated. Specifically, starting from previously reported lead compounds, peripheral substitution patterns of a series of biaryl-linked pyrrolidine NS5A replication complex inhibitors were probed and structure-activity relationships were elucidated. Using molecular modelling and a supercritical fluid chromatographic (SFC) technique, intramolecular H-bonding and peripheral functional group topology were evaluated as key determinants of activity and membrane permeability. The novel compounds exhibited retained potency as compared with the lead compounds, and also showed promising results against a panel of resistance viruses. Together, the results of the study take us a step closer towards understanding the potency of daclatasvir, a clinical candidate upon which the compounds were based, and to designing improved analogues as second-generation antiviral agents targeting NS5A.

Small-molecule inhibitors of the LEDGF/p75 binding site of integrase block HIV replication and modulate integrase multimerization.

Targeting the HIV integrase (HIV IN) is a clinically validated approach for designing novel anti-HIV therapies. We have previously described the discovery of a novel class of integration inhibitors, 2-(quinolin-3-yl)acetic acid derivatives, blocking HIV replication at a low micromolar concentration through binding in the LEDGF/p75 binding pocket of HIV integrase, hence referred to as LEDGINs. Here we report the detailed characterization of their mode of action. The design of novel and more potent analogues with nanomolar activity enabled full virological evaluation and a profound mechanistic study. As allosteric inhibitors, LEDGINs bind to the LEDGF/p75 binding pocket in integrase, thereby blocking the interaction with LEDGF/p75 and interfering indirectly with the catalytic activity of integrase. Detailed mechanism-of-action studies reveal that the allosteric mode of inhibition is likely caused by an effect on HIV-1 integrase oligomerization. The multimodal inhibition by LEDGINs results in a block in HIV integration and in a replication deficiency of progeny virus. The allosteric nature of LEDGINs leads to synergy in combination with the clinically approved active site HIV IN strand transfer inhibitor (INSTI) raltegravir, and cross-resistance profiling proves the distinct mode of action of LEDGINs and INSTIs. The allosteric nature of inhibition and compatibility with INSTIs underline an interest in further (clinical) development of LEDGINs.

Colony-forming assays reveal enhanced suppression of hepatitis C virus replication using combinations of direct-acting antivirals.

The current standard of care for patients infected with hepatitis C virus (HCV) is not effective universally and is associated with severe side effects. Direct-acting antiviral molecules have potential to transform treatment of HCV-infected individuals but emergence of drug-resistant virus will be problematic. It is anticipated that, to limit the emergence of drug-resistant virus, future HCV therapies must consist of multiple direct-acting antivirals. In the present study, cell culture-based colony-forming assays were used to demonstrate enhanced suppression of HCV RNA replication following simultaneous treatment of HCV replicon-containing cells with two direct-acting antivirals. Specifically, combinations of NS5Ai and Filibuvir (small molecule inhibitors of HCV-encoded NS5A and NS5B proteins respectively) were able to suppress colony formation fully at concentrations that individually they could not. HCV replicon RNA isolated from colonies that emerged following treatment with suboptimal concentrations of NS5Ai were found to encode resistance substitutions in the NS5A gene, which rendered them insensitive to subsequent high doses of NS5Ai. Furthermore, both NS5Ai and Filibuvir were effective at suppressing colony formation in combination with BILN 2061, an inhibitor of HCV-encoded NS3. Collectively, these data underscore the increased inhibitory capacity of direct-acting antivirals to suppress HCV RNA replication when present in combination.

Small molecules targeting hepatitis C virus-encoded NS5A cause subcellular redistribution of their target: insights into compound modes of action.

The current standard of care for hepatitis C virus (HCV)-infected patients consists of lengthy treatment with interferon and ribavirin. To increase the effectiveness of HCV therapy, future regimens will incorporate multiple direct-acting antiviral (DAA) drugs. Recently, the HCV-encoded NS5A protein has emerged as a promising DAA target. Compounds targeting NS5A exhibit remarkable potency in vitro and demonstrate early clinical promise, suggesting that NS5A inhibitors could feature in future DAA combination therapies. Since the mechanisms through which these molecules operate are unknown, we have used NS5A inhibitors as tools to investigate their modes of action. Analysis of replicon-containing cells revealed dramatic phenotypic alterations in NS5A localization following treatment with NS5A inhibitors; NS5A was redistributed from the endoplasmic reticulum to lipid droplets. The NS5A relocalization did not occur in cells treated with other classes of HCV inhibitors, and NS5A-targeting molecules did not cause similar alterations in the localization of other HCV-encoded proteins. Time course analysis of the redistribution of NS5A revealed that the transfer of protein to lipid droplets was concomitant with the onset of inhibition, as judged by the kinetic profiles for these compounds. Furthermore, analysis of the kinetic profile of inhibition for a panel of test molecules permitted the separation of compounds into different kinetic classes based on their modes of action. Results from this approach suggested that NS5A inhibitors perturbed the function of new replication complexes, rather than acting on preformed complexes. Taken together, our data reveal novel biological consequences of NS5A inhibition, which may help enable the development of future assay platforms for the identification of new and/or different NS5A inhibitors.

Selection, optimization, and pharmacokinetic properties of a novel, potent antiviral locked nucleic acid-based antisense oligomer targeting hepatitis C virus internal ribosome entry site.

We have screened 47 locked nucleic acid (LNA) antisense oligonucleotides (ASOs) targeting conserved (>95% homology) sequences in the hepatitis C virus (HCV) genome using the subgenomic HCV replicon assay and generated both antiviral (50% effective concentration [EC(50)]) and cytotoxic (50% cytotoxic concentration [CC(50)]) dose-response curves to allow measurement of the selectivity index (SI). This comprehensive approach has identified an LNA ASO with potent antiviral activity (EC(50) = 4 nM) and low cytotoxicity (CC(50) >880 nM) targeting the 25- to 40-nucleotide region (nt) of the HCV internal ribosome entry site (IRES) containing the distal and proximal miR-122 binding sites. LNA ASOs targeting previously known accessible regions of the IRES, namely, loop III and the initiation codon in loop IV, had poor SI values. We optimized the LNA ASO sequence by performing a 1-nucleotide walk through the 25- to 40-nt region and show that the boundaries for antiviral efficacy are extremely precise. Furthermore, we have optimized the format for the LNA ASO using different gapmer and mixomer patterns and show that RNase H is required for antiviral activity. We demonstrate that RNase H-refractory ASOs targeting the 25- to 40-nt region have no antiviral effect, revealing important regulatory features of the 25- to 40-nt region and suggesting that RNase H-refractory LNA ASOs can act as potential surrogates for proviral functions of miR-122. We confirm the antisense mechanism of action using mismatched LNA ASOs. Finally, we have performed pharmacokinetic experiments to demonstrate that the LNA ASOs have a very long half-life (>5 days) and attain hepatic maximum concentrations >100 times the concentration required for in vitro antiviral activity.

HIV capsid is a tractable target for small molecule therapeutic intervention.

Despite a high current standard of care in antiretroviral therapy for HIV, multidrug-resistant strains continue to emerge, underscoring the need for additional novel mechanism inhibitors that will offer expanded therapeutic options in the clinic. We report a new class of small molecule antiretroviral compounds that directly target HIV-1 capsid (CA) via a novel mechanism of action. The compounds exhibit potent antiviral activity against HIV-1 laboratory strains, clinical isolates, and HIV-2, and inhibit both early and late events in the viral replication cycle. We present mechanistic studies indicating that these early and late activities result from the compound affecting viral uncoating and assembly, respectively. We show that amino acid substitutions in the N-terminal domain of HIV-1 CA are sufficient to confer resistance to this class of compounds, identifying CA as the target in infected cells. A high-resolution co-crystal structure of the compound bound to HIV-1 CA reveals a novel binding pocket in the N-terminal domain of the protein. Our data demonstrate that broad-spectrum antiviral activity can be achieved by targeting this new binding site and reveal HIV CA as a tractable drug target for HIV therapy.

A low-molecular-weight entry inhibitor of both CCR5- and CXCR4-tropic strains of human immunodeficiency virus type 1 targets a novel site on gp41.

A low-molecular-weight human immunodeficiency virus type 1 (HIV-1) inhibitor, PF-68742 (molecular weight, 573), has been identified in a high-throughput screen for compounds that block HIV-1 envelope glycoprotein (Env)-mediated fusion. The compound is shown to be potent against R5 and X4 isolates in both cell-cell fusion and antiviral assays (50% effective concentrations of approximately 0.1 to 1 muM). Postfusion and HIV-1 pseudotyping control experiments confirm that PF-68742 is an entry inhibitor with Env as the specific target for antiviral action. PF-68742 was not able to block binding of monomeric gp120 to soluble CD4 or the binding of gp120:CD4 complexes to cell-associated CCR5, thus distinguishing PF-68742 from described gp120 antagonists and coreceptor binders. Escape variants of HIV-1(NL4-3) were selected, and all resistant viruses were found to contain a common G514R (HxB2 numbering) mutation in Env, located proximal to the furin cleavage site in the fusion peptide of gp41. When introduced into wild-type NL4-3 gp41, G514R conferred resistance to PF-68742. Resistance via G514R is shown to be associated with enhancement of virion infectivity by PF-68742 that may result from altered properties of inhibitor-bound Env, rather than from a loss of compound binding. Wild-type viruses and those with substitutions in the disulfide loop (DSL) region of gp41 were also examined for PF-68742 sensitivity. Here, complete resistance to PF-68742 was found to occur through changes outside of position 514, including in the gp41 DSL region. The results highlight PF-68742 as a starting point for novel therapies against HIV-1 and provide new insights into models of Env-mediated fusion.

New small-molecule inhibitor class targeting human immunodeficiency virus type 1 virion maturation.

A new small-molecule inhibitor class that targets virion maturation was identified from a human immunodeficiency virus type 1 (HIV-1) antiviral screen. PF-46396, a representative molecule, exhibits antiviral activity against HIV-1 laboratory strains and clinical isolates in T-cell lines and peripheral blood mononuclear cells (PBMCs). PF-46396 specifically inhibits the processing of capsid (CA)/spacer peptide 1 (SP1) (p25), resulting in the accumulation of CA/SP1 (p25) precursor proteins and blocked maturation of the viral core particle. Viral variants resistant to PF-46396 contain a single amino acid substitution in HIV-1 CA sequences (CAI201V), distal to the CA/SP1 cleavage site in the primary structure, which we demonstrate is sufficient to confer significant resistance to PF-46396 and 3-O-(3',3'-dimethylsuccinyl) betulinic acid (DSB), a previously described maturation inhibitor. Conversely, a single amino substitution in SP1 (SP1A1V), which was previously associated with DSB in vitro resistance, was sufficient to confer resistance to DSB and PF-46396. Further, the CAI201V substitution restored CA/SP1 processing in HIV-1-infected cells treated with PF-46396 or DSB. Our results demonstrate that PF-46396 acts through a mechanism that is similar to DSB to inhibit the maturation of HIV-1 virions. To our knowledge, PF-46396 represents the first small-molecule HIV-1 maturation inhibitor that is distinct in chemical class from betulinic acid-derived maturation inhibitors (e.g., DSB), demonstrating that molecules of diverse chemical classes can inhibit this mechanism.

Determination of 238u/235u, 236u/238u and uranium concentration in urine using sf-icp-ms and mc-icp-ms: an interlaboratory comparison.

Accidental exposure to depleted or enriched uranium may occur in a variety of circumstances. There is a need to quantify such exposure, with the possibility that the testing may post-date exposure by months or years. Therefore, it is important to develop a very sensitive test to measure precisely the isotopic composition of uranium in urine at low levels of concentration. The results of an interlaboratory comparison using sector field (SF)-inductively coupled plasma-mass spectrometry (ICP-MS) and multiple collector (MC)-ICP-MS for the measurement of uranium concentration and U/U and U/U isotopic ratios of human urine samples are presented. Three urine samples were verified to contain uranium at 1-5 ng L and shown to have natural uranium isotopic composition. Portions of these urine batches were doped with depleted uranium (DU) containing small quantities of U, and the solutions were split into 100 mL and 400 mL aliquots that were subsequently measured blind by three laboratories. All methods investigated were able to measure accurately U/U with precisions of approximately 0.5% to approximately 4%, but only selected MC-ICP-MS methods were capable of consistently analyzing U/U to reasonable precision at the approximately 20 fg L level of U abundance. Isotope dilution using a U tracer demonstrates the ability to measure concentrations to better than +/-4% with the MC-ICP-MS method, though sample heterogeneity in urine samples was shown to be problematic in some cases. MC-ICP-MS outperformed SF-ICP-MS methods, as was expected. The MC-ICP-MS methodology described is capable of measuring to approximately 1% precision the U/U of any sample of human urine over the entire range of uranium abundance down to <1 ng L, and detecting very small amounts of DU contained therein.