Preclinical characterization of a non-peptidomimetic HIV protease inhibitor with improved metabolic stability.

Protease inhibitors (PIs) remain an important component of antiretroviral therapy for the treatment of HIV-1 infection due to their high genetic barrier to resistance development. Nevertheless, the two most commonly prescribed HIV PIs, atazanavir and darunavir, still require co-administration with a pharmacokinetic boosting agent to maintain sufficient drug plasma levels which can lead to undesirable drug-drug interactions. Herein, we describe GS-9770, a novel investigational non-peptidomimetic HIV PI with unboosted once-daily oral dosing potential due to improvements in its metabolic stability and its pharmacokinetic properties in preclinical animal species. This compound demonstrates potent inhibitory activity and high on-target selectivity for recombinant HIV-1 protease versus other aspartic proteases tested. In cell culture, GS-9770 inhibits Gag polyprotein cleavage and shows nanomolar anti-HIV-1 potency in primary human cells permissive to HIV-1 infection and against a broad range of HIV subtypes. GS-9770 demonstrates an improved resistance profile against a panel of patient-derived HIV-1 isolates with resistance to atazanavir and darunavir. In resistance selection experiments, GS-9770 prevented the emergence of breakthrough HIV-1 variants at all fixed drug concentrations tested and required multiple protease substitutions to enable outgrowth of virus exposed to escalating concentrations of GS-9770. This compound also remained fully active against viruses resistant to drugs from other antiviral classes and showed no in vitro antagonism when combined pairwise with drugs from other antiretroviral classes. Collectively, these preclinical data identify GS-9770 as a potent, non-peptidomimetic once-daily oral HIV PI with potential to overcome the persistent requirement for pharmacological boosting with this class of antiretroviral agents.

The Single-Component Flavin Reductase/Flavin-Dependent Halogenase AetF is a Versatile Catalyst for Selective Bromination and Iodination of Arenes and Olefins.

Flavin-dependent halogenases (FDHs) natively catalyze selective halogenation of electron rich aromatic and enolate groups. Nearly all FDHs reported to date require a separate flavin reductase to supply them with FADH2 , which complicates biocatalysis applications. In this study, we establish that the single component flavin reductase/flavin dependent halogenase AetF catalyzes halogenation of a diverse set of substrates using a commercially available glucose dehydrogenase to drive its halogenase activity. High site selectivity, activity on relatively unactivated substrates, and high enantioselectivity for atroposelective bromination and bromolactonization was demonstrated. Site-selective iodination and enantioselective cycloiodoetherification was also possible using AetF. The substrate and reaction scope of AetF suggest that it has the potential to greatly improve the utility of biocatalytic halogenation.

Color, structure, and rheology of a diblock bottlebrush copolymer solution.

A structure-property-process relation is established for a diblock bottlebrush copolymer solution, through a combination of rheo-neutron scattering, imaging, and rheological measurements. Polylactic acid-b-polystyrene diblock bottlebrush copolymers were dispersed in toluene with a concentration of 175 mg ml-1, where they self-assembled into a lamellar phase. All measurements were carried out at 5 °C. The solution color, as observed in reflection, is shown to be a function of the shear rate. Under equilibrium and near-equilibrium conditions, the solution has a green color. At low shear rates the solution remains green, while at intermediate rates the solution is cyan. At the highest rates applied the solution is indigo. The lamellar spacing is shown to be a decreasing function of shear rate, partially accounting for the color change. The lamellae are oriented 'face-on' with the wall under quiescence and low shear rates, while a switch to 'edge-on' is observed at the highest shear rates, where the reflected color disappears. The intramolecular distance between bottlebrush polymers does not change with shear rate, although at high shear rates, the bottlebrush polymers are preferentially aligned in the vorticity direction within the lamellae. We therefore form a consistent relation between structure and function, spanning a wide range of length scales and shear rates.

Redox-Neutral TEMPO Catalysis: Direct Radical (Hetero)Aryl C-H Di- and Trifluoromethoxylation.

Applications of TEMPO. catalysis for the development of redox-neutral transformations are rare. Reported here is the first TEMPO. -catalyzed, redox-neutral C-H di- and trifluoromethoxylation of (hetero)arenes. The reaction exhibits a broad substrate scope, has high functional-group tolerance, and can be employed for the late-stage functionalization of complex druglike molecules. Kinetic measurements, isolation and resubjection of catalytic intermediates, UV/Vis studies, and DFT calculations support the proposed oxidative TEMPO. /TEMPO+ redox catalytic cycle. Mechanistic studies also suggest that Li2 CO3 plays an important role in preventing catalyst deactivation. These findings will provide new insights into the design and development of novel reactions through redox-neutral TEMPO. catalysis.

Structure-Property Relationships via Recovery Rheology in Viscoelastic Materials.

The recoverable strain is shown to correlate to the temporal evolution of microstructure via time-resolved small-angle neutron scattering and dynamic shear rheology. Investigating two distinct polymeric materials of wormlike micelles and fibrin network, we demonstrate that, in addition to the nonlinear structure-property relationships, the shear and normal stress evolution is dictated by the recoverable strain. A distinct sequence of physical processes under large amplitude oscillatory shear (LAOS) is identified that clearly contains information regarding both the steady-state flow curve and the linear-regime frequency sweep, contrary to most interpretations that LAOS responses are either distinct from or somehow intermediate between the two cases. This work provides a physically motivated and straightforward path to further explore the structure-property relationships of viscoelastic materials under dynamic flow conditions.

Rheological Analysis of the Gelation Kinetics of an Enzyme Cross-linked PEG Hydrogel.

The diverse requirements of hydrogels for tissue engineering motivate the development of cross-linking reactions to fabricate hydrogel networks with specific features, particularly those amenable to the activity of biological materials (e.g., cells, proteins) that do not require exposure to UV light. We describe gelation kinetics for a library of thiolated poly(ethylene glycol) sulfhydryl hydrogels undergoing enzymatic cross-linking via horseradish peroxidase, a catalyst-driven reaction activated by hydrogen peroxide. We report the use of small-amplitude oscillatory shear (SAOS) to quantify gelation kinetics as a function of reaction conditions (hydrogen peroxide and polymer concentrations). We employ a novel approach to monitor the change of viscoelastic properties of hydrogels over the course of gelation (Δ tgel) via the time derivative of the storage modulus (d G'/d t). This approach, fundamentally distinct from traditional methods for defining a gel point, quantifies the time interval over which gelation events occur. We report that gelation depends on peroxide and polymer concentrations as well as system temperature, where the effects of hydrogen peroxide tend to saturate over a critical concentration. Further, this cross-linking reaction can be reversed using l-cysteine for rapid cell isolation, and the rate of hydrogel dissolution can be monitored using SAOS.

Strain shifts under stress-controlled oscillatory shearing in theoretical, experimental, and structural perspectives: Application to probing zero-shear viscosity.

Rheological measurements in which the applied stress or strain is oscillated are widely used to interrogate viscoelastic properties due to the independent control over the time scale and length scale afforded by changes in amplitude and frequency. Taking a nontraditional approach, we treat stress-controlled oscillatory tests as creep tests with transiently varying stress and apply an analysis typically used for steady creep and recovery experiments. Defining zero strain as the state prior to external shearing, it is shown that strain responses to small-amplitude oscillatory stressing are naturally shifted from the starting point by an amount proportional to the phase of the applied stress. The phenomenology is experimentally observed with entangled polymerlike micelles and polyethylene oxide solutions. A theory of strain shifting in the steady alternating state is provided based on recovery rheology, where differences between total strain and recoverable strains are acknowledged. User-controlled variables, such as the amplitude of the stress, the angular frequency, and the phase of the stress, as well a lone material parameter, the zero-shear viscosity, are shown to dictate the amount of shifting. A rapid and efficient approach of determining the zero-shear viscosity is, therefore, presented. We investigate the microstructural evolution via in situ small-angle neutron scattering when strain shifting appears. The microscopic orientation is shown to correlate to the recoverable strain independent of the shifting. Additional measurements are carried out on collagen, pluronic-hyaluronic acid, alginate gels, and polystyrene melts to show the generic nature of the strain shift phenomenon. In addition, we demonstrate that the strain-shift knowledge can be applied to determine the horizontal shift factor in time-temperature superposition, free of any numerical fitting procedures.

Synthesis of Tri- and Difluoromethoxylated Compounds by Visible-Light Photoredox Catalysis.

Trifluoromethoxy (OCF3 ) and difluoromethoxy (OCF2 H) groups are fluorinated structural motifs that exhibit unique physicochemical characteristics. Incorporation of these substituents into organic molecules is a highly desirable approach used in medicinal chemistry and drug discovery processes to alter the properties of a parent compound. Recently, tri- and difluoromethyl ethers have received increasing attention and several innovative strategies to access these valuable functional groups have been developed. The focus of this Minireview is the use of visible-light photoredox catalysis in the synthesis of tri- and difluoromethyl ethers. Recent photocatalytic strategies for the formation of O-CF3 , C-OCF3, O-CF2 H, and C-OCF2 H bonds as well as other transformations leading to the construction of ORF groups are discussed herein.

Recent Development of Catalytic Trifluoromethoxylation Reactions.

Trifluoromethoxy (OCF3) group exhibits unique properties, which render it a highly desirable structural motif in life and materials sciences. The numbers of newly synthesized trifluoromethyl ethers are booming as new synthetic methods are constantly being developed to access these valuable compounds. This Review summarizes recent catalytic approaches towards preparation of trifluoromethyl ethers. Alkene, allylic, benzylic, and aryl trifluoromethoxylation reactions are discussed herein.

Catalytic C-H Trifluoromethoxylation of Arenes and Heteroarenes.

The intermolecular C-H trifluoromethoxylation of arenes remains a long-standing and unsolved problem in organic synthesis. Herein, we report the first catalytic protocol employing a novel trifluoromethoxylating reagent and redox-active catalysts for the direct (hetero)aryl C-H trifluoromethoxylation. Our approach is operationally simple, proceeds at room temperature, uses easy-to-handle reagents, requires only 0.03 mol % of redox-active catalysts, does not need specialized reaction apparatus, and tolerates a wide variety of functional groups and complex structures such as sugars and natural product derivatives. Importantly, both ground-state and photoexcited redox-active catalysts are effective. Detailed computational and experimental studies suggest a unique reaction pathway where photoexcitation of the trifluoromethoxylating reagent releases the OCF3 radical that is trapped by (hetero)arenes. The resulting cyclohexadienyl radicals are oxidized by redox-active catalysts and deprotonated to form the desired products of trifluoromethoxylation.

Redox-Active Reagents for Photocatalytic Generation of the OCF3 Radical and (Hetero)Aryl C-H Trifluoromethoxylation.

The trifluoromethoxy (OCF3 ) radical is of great importance in organic chemistry. Yet, the catalytic and selective generation of this radical at room temperature and pressure remains a longstanding challenge. Herein, the design and development of a redox-active cationic reagent (1) that enables the formation of the OCF3 radical in a controllable, selective, and catalytic fashion under visible-light photocatalytic conditions is reported. More importantly, the reagent allows catalytic, intermolecular C-H trifluoromethoxylation of a broad array of (hetero)arenes and biorelevant compounds. Experimental and computational studies suggest single electron transfer (SET) from excited photoredox catalysts to 1 resulting in exclusive liberation of the OCF3 radical. Addition of this radical to (hetero)arenes gives trifluoromethoxylated cyclohexadienyl radicals that are oxidized and deprotonated to afford the products of trifluoromethoxylation.

Aspirin use associated with amyotrophic lateral sclerosis: a total population-based case-control study.

BACKGROUND: The association of aspirin use and nonsteroid anti-inflammatory drug (NSAID) use with amyotrophic lateral sclerosis (ALS) risk is unclear. This study determined whether use of any individual compound is associated with ALS risk by conducting a total population-based case-control study in Taiwan. METHODS: A total of 729 patients with newly diagnosed ALS who had a severely disabling disease certificate between January 1, 2002, and December 1, 2008, comprised the case group. These cases were compared with 7290 sex-, age-, residence-, and insurance premium-matched controls. Drug use by each Anatomical Therapeutic Chemical code was analyzed using conditional logistic regression models. False discovery rate (FDR)-adjusted P values were reported in order to avoid inflating false positives. RESULTS: Of the 1336 compounds, only the 266 with use cases exceeding 30 in our database were included in the screening analysis. Without controlling for steroid use, the analysis failed to reveal any compound that was inversely associated with ALS risk according to FDR criteria. After controlling for steroid use, we found use of the following compounds to be associated with ALS risk: aspirin, diphenhydramine (one of the antihistamines), and mefenamic acid (one of the NSAIDs). A multivariate analysis revealed that aspirin was independently inversely associated with ALS risk after controlling for diphenhydramine, mefenamic acid, and steroid use. The inverse association between aspirin and ALS was present predominately in patients older than 55 years. CONCLUSIONS: The results of this study suggested that aspirin use might reduce the risk of ALS, and the benefit might be more prominent for older people.

Liposomal bupivacaine interscalene blocks demonstrate a greater proportion of total shoulder arthroplasty patients with clinically tolerable pain: a retrospective quality improvement study of 491 patients.

OBJECTIVE: To evaluate the effects of liposomal bupivacaine use for interscalene blocks on postoperative analgesia in total shoulder arthroplasty patients. METHODS: De-identified total or reverse total shoulder arthroplasty patients between 2018 and 2021 were analyzed. Patients were grouped into single shot interscalene block with liposomal bupivacaine (LB) with plain bupivacaine, other block (OB) with other local anesthetics (mepivacaine, ropivacaine, or plain bupivacaine), or no block (NB). The primary outcome was the proportion of patients with clinically tolerable pain scores (mean VAS ≤4) from 0 to 24 â€‹h in each group. Secondary outcomes included averaged visual analog pain scores (VAS) and opioid consumption measured in morphine milligram equivalents (MMEs) from 0 to 24 â€‹h. We also analyzed the proportion of patients with clinically tolerable pain, mean VAS, and opioid consumption from 0 to 72 â€‹h in those patients with at least a 3-day hospital length of stay. RESULTS: A total of 491 de-identified total shoulder arthroplasty patients, 285 liposomal bupivacaine group (LB), 178 other block group (OB), and 28 no block group (NB), were analyzed. The primary outcome showed a statistically significant different proportion of patients with clinically tolerable pain from 0 to 24 â€‹h in the LB group (69 â€‹%) vs. OB group (39 â€‹%) vs. NB group (11 â€‹%) (<0.001). Secondary outcomes included statistically significant differences in VAS (LB median â€‹= â€‹3.35, OB median â€‹= â€‹4.38, NB median â€‹= â€‹5.25 (p â€‹< â€‹0.001, <0.001)) and total MME opioid consumption (LB median â€‹= â€‹40, OB median â€‹= â€‹60, NB median â€‹= â€‹88 (p â€‹< â€‹0.001, 0.001)) between groups from 0 to 24 â€‹h. For patients who had hospital stays of at least 3 days, a significant association was found with having achieved clinically tolerable pain 0-72 â€‹h and the LB group (51 â€‹%) vs. OB group (21 â€‹%) vs. NB group (11 â€‹%) (P â€‹= â€‹0.006). However, there was no statistical difference in mean VAS or opioid consumption between these groups. CONCLUSION: A greater proportion of total shoulder arthroplasty patients that received liposomal bupivacaine in interscalene block have clinically tolerable pain scores from 0 to 24 â€‹h, lower VAS, and lower MME consumption in patients following total shoulder arthroplasty. LEVEL OF EVIDENCE: Level III - Clinical Study.

Arenavirus-Based Vectors Generate Robust SIV Immunity in Non-Human Primates.

Arenavirus-based vectors are being investigated as therapeutic vaccine candidates with the potential to elicit robust CD8 T-cell responses. We compared the immunogenicity of replicating (artPICV and artLCMV) and non-replicating (rPICV and rLCMV) arenavirus-based vectors expressing simian immunodeficiency virus (SIV) Gag and Envelope (Env) immunogens in treatment-naïve non-human primates. Heterologous regimens with non-replicating and replicating vectors elicited more robust SIV IFN-γ responses than a homologous regimen, and replicating vectors elicited significantly higher cellular immunogenicity than non-replicating vectors. The heterologous regimen elicited high anti-Env antibody titers when administered intravenously, with replicating vectors inducing significantly higher titers than non-replicating vectors. Intramuscular immunization resulted in more durable antibody responses than intravenous immunization for both vector platforms, with no difference between the replicating and non-replicating vectors. Overall, both replicating and non-replicating arenavirus vectors generated robust T- and B-cell-mediated immunity to SIV antigens in treatment-naïve non-human primates, supporting further evaluation of these vectors in a clinical setting for HIV therapy.

Cellular responses to L-serine in Saccharomyces cerevisiae: roles of general amino acid control, compartmentalization, and aspartate synthesis.

In addition to its other roles, L-serine functions in one-carbon metabolism and is interconvertable with glycine via serine hydroxymethyltransferases. However, the transcriptional response by Saccharomyces cerevisiae to L-serine addition is markedly different from that to glycine, with L-serine acting as a nutrient source rather than one-carbon units. Following addition of excess L-serine, 743 genes showed significant expression changes. Induced functions included amino acid synthesis, some stress responses, and FeS metabolism, while ribosomal RNA processing, ribosome biogenesis and hexose transport were repressed. A co-regulated network of ten transcription factors could together control more than 90% of the induced and repressed genes forming a general response to changes induced by other amino acids or stresses and including the general amino acid control system usually activated in response to starvation for amino acids. A specific response to L-serine was induction of CHA1 encoding serine (threonine) dehydratase. L-serine addition resulted in a substantial transient increase in L-aspartate, which is, rather than L-glutamate, the major metabolite for short-term storage of ammonia derived from degradation of L-serine. L-aspartate synthesis was exclusively through mitochondrial metabolism of L-serine to pyruvate and ammonia, involving Cha1p, cytoplasmic pyruvate carboxylases Pyc1p and Pyc2p, and the cytoplasmic aspartate aminotransferase Aat2p.

A global spatial analysis of factors associated with case and mortality rates for coronavirus disease 2019 during the first year of the pandemic.

BACKGROUND: A increasing number of studies have revealed associations between country-level determinants and coronavirus disease 2019 (COVID-19) outcomes. This ecological study was conducted to analyze country-level parameters related to COVID-19 infections and deaths during the first year of the pandemic. METHODS: The examined predictors comprised demographics, economic factors, disease prevalence and healthcare system status, and the relevant data were obtained from public databases. The index dates were set to 15 July 2020 (Time 1) and 15 December 2020 (Time 2). The adjusted spatial autoregression models used a first-order queen contiguity spatial weight for the main analysis and a second-order queen contiguity spatial weight for a sensitivity analysis to examine the predictors associated with COVID-19 case and mortality rates. RESULTS: Obesity was significantly and positively associated with COVID-19 case and mortality rates in both the main and sensitivity analyses. The sensitivity analysis revealed that a country's gross domestic product, population density, life expectancy and proportion of the population older than 65 y are positively associated with COVID-19 case and mortality rates. CONCLUSIONS: With the increasing global prevalence of obesity, the relationship between obesity and COVID-19 disease at the country level must be clarified and continually monitored.

Substance use before or during pregnancy and the risk of child mortality, perinatal morbidities and congenital anomalies.

AIMS: We aimed to investigate child mortality, perinatal morbidities and congenital anomalies born by women with substance misuse during or before pregnancy (DP or BP). METHODS: Taiwan Birth Registration from 2004 to 2014 linking Integrated Illicit Drug Databases used to include substance misuse participates. Children born by mothers convicted of substance misuse DP or BP were the substance-exposed cohort. Two substance-unexposed comparison cohorts were established: one comparison cohort selected newborns from the rest of the population on a ratio of 1:1 and exact matched by the child's gender, child's birth year, mother's birth year and child's first use of the health insurance card; another comparison cohort matched newborns from exposed and unexposed mothers by their propensity scores calculated from logistic regression. RESULTS: The exposure group included 1776 DP, 1776 BP and 3552 unexposed individuals in exact-matched cohorts. A fourfold increased risk of deaths in children born by mothers exposed to substance during pregnancy was found compared to unexposed group (hazard ratio [HR] = 4.54, 95% confidence interval (CI): 2.07-9.97]. Further multivariate Cox regression models with adjustments and propensity matching substantially attenuated HRs on mortality in the substance-exposed cohort (aHR = 1.62, 95% CI: 1.10-2.39). Raised risks of perinatal morbidities and congenital anomalies were also found. CONCLUSIONS: Increased risks of child mortality, perinatal morbidities or congenital anomalies were found in women with substance use during pregnancy. From estimates before and after adjustments, our results showed that having outpatient visits or medical utilizations during pregnancy were associated with substantially attenuated HRs on mortality in the substance-exposed cohort. Therefore, the excess mortality risk might be partially explained by the lack of relevant antenatal clinical care. Our finding may suggest that the importance of early identification, specific abstinence program and access to appropriate antenatal care might be helpful in reducing newborn mortality. Adequate prevention policies may be formulated.

Development of the Commercial Manufacturing Process for Nirmatrelvir in 17 Months.

The advancement of nirmatrelvir, the active ingredient in Paxlovid, from discovery to emergency use authorization was achieved in just 17 months, requiring an unprecedented rate of chemical process development.

Immunogenic arenavirus vector SIV vaccine reduces setpoint viral load in SIV-challenged rhesus monkeys.

HIV affects more than 38 million people worldwide. Although HIV can be effectively treated by lifelong combination antiretroviral therapy, only a handful of patients have been cured. Therapeutic vaccines that induce robust de novo immune responses targeting HIV proteins and latent reservoirs will likely be integral for functional HIV cure. Our study shows that immunization of naïve rhesus macaques with arenavirus-derived vaccine vectors encoding simian immunodeficiency virus (SIVSME543 Gag, Env, and Pol) immunogens is safe, immunogenic, and efficacious. Immunization induced robust SIV-specific CD8+ and CD4+ T-cell responses with expanded cellular breadth, polyfunctionality, and Env-binding antibodies with antibody-dependent cellular cytotoxicity. Vaccinated animals had significant reductions in median SIV viral load (1.45-log10 copies/mL) after SIVMAC251 challenge compared with placebo. Peak viral control correlated with the breadth of Gag-specific T cells and tier 1 neutralizing antibodies. These results support clinical investigation of arenavirus-based vectors as a central component of therapeutic vaccination for HIV cure.

Tri-substituted acylhydrazines as tertiary amide bioisosteres: HCV NS5B polymerase inhibitors.

The use of a tri-substituted acylhydrazine as an isostere of a tertiary amide was explored in a series of HCV NS5B thumb site II inhibitors. Direct replacement generated an analog with similar conformational and physicochemical properties. The series was extended to produce compounds with potent binding affinities and encouraging levels of cellular potency.