Engineering Hydroxylase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of a Key Intermediate to Belzutifan.

Biocatalytic oxidations are an emerging technology for selective C-H bond activation. While promising for a range of selective oxidations, practical use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering and practical application of a non-heme iron and α-ketoglutarate-dependent dioxygenase for the direct stereo- and regio-selective hydroxylation of a non-native fluoroindanone en route to the oncology treatment belzutifan, replacing a five-step chemical synthesis with a direct enantioselective hydroxylation. Mechanistic studies indicated that formation of the desired product was limited by enzyme stability and product overoxidation, with these properties subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. Highlighting the industrial utility of this biocatalyst, the high-yielding, green, and efficient oxidation was demonstrated at kilogram scale for the synthesis of belzutifan.

Sexual Mixing by HIV Status and Pre-exposure Prophylaxis Use Among Men Who Have Sex With Men: Addressing Information Bias.

BACKGROUND: Population-level estimates of sexual network mixing for parameterizing prediction models of pre-exposure prophylaxis (PrEP) effectiveness are needed to inform prevention of HIV transmission among men who have sex with men (MSM). Estimates obtained by egocentric sampling are vulnerable to information bias due to incomplete respondent knowledge. METHODS: We estimated patterns of serosorting and PrEP sorting among MSM in the United States using data from a 2017-2019 egocentric sexual network study. Respondents served as proxies to report the HIV status and PrEP use of recent sexual partners. We contrasted results from a complete-case analysis (unknown HIV and PrEP excluded) versus a bias analysis with respondent-reported data stochastically reclassified to simulate unobserved self-reported data from sexual partners. RESULTS: We found strong evidence of preferential partnering across analytical approaches. The bias analysis showed concordance between sexual partners of HIV diagnosis and PrEP use statuses for MSM with diagnosed HIV (39%; 95% simulation interval: 31, 46), MSM who used PrEP (32%; 21, 37), and MSM who did not use PrEP (83%; 79, 87). The fraction of partners with diagnosed HIV was higher among MSM who used PrEP (11%; 9, 14) compared with MSM who did not use PrEP (4%; 3, 5). Comparatively, across all strata of respondents, the complete-case analysis overestimated the fractions of partners with diagnosed HIV or PrEP use. CONCLUSIONS: We found evidence consistent with HIV and PrEP sorting among MSM, which may decrease the population-level effectiveness of PrEP. Bias analyses can improve mixing estimates for parameterization of transmission models.

Projected Impact of Concurrently Available Long-Acting Injectable and Daily-Oral Human Immunodeficiency Virus Preexposure Prophylaxis: A Mathematical Model.

BACKGROUND: Long-acting injectable (LAI) human immunodeficiency virus (HIV) preexposure prophylaxis (PrEP) is reportedly efficacious, although full trial results have not been published. We used a dynamic network model of HIV transmission among men who have sex with men to assess the population impact of LAI-PrEP when available concurrently with daily-oral (DO) PrEP. METHODS: The reference model represents the current HIV epidemiology and DO-PrEP coverage (15% among those with behavioral indications for PrEP) among men who have sex with men in the southeastern United States. Primary analyses investigated varied PrEP uptake and proportion selecting LAI-PrEP. Secondary analyses evaluated uncertainty in pharmacokinetic efficacy and LAI-PrEP persistence relative to DO-PrEP. RESULTS: Compared with the reference scenario, if 50% chose LAI-PrEP, 4.3% (95% simulation interval, -7.3% to 14.5%) of infections would be averted over 10 years. The impact of LAI-PrEP is slightly greater than that of the DO-PrEP-only regimen, based on assumptions of higher adherence and partial protection after discontinuation. If the total PrEP initiation rate doubled, 17.1% (95% simulation interval, 6.7%-26.4%) of infections would be averted. The highest population-level impact occurred when LAI-PrEP uptake and persistence improved. CONCLUSIONS: If LAI-PrEP replaces DO-PrEP, its availability will modestly improve the population impact. LAI-PrEP will make a more substantial impact if its availability drives higher total PrEP coverage, or if persistence is greater for LAI-PrEP.

A Behavioral Cascade of HIV Seroadaptation Among US Men Who Have Sex with Men in the Era of PrEP and U = U.

Seroadaptive behaviors help reduce HIV risk for some men who have sex with men (MSM), and have been well documented across MSM populations. Advancements in biomedical prevention have changed the contexts in which seroadaptive behaviors occur. We thus sought to estimate and compare the prevalence of four stages of the "seroadaptive cascade" by PrEP use in the recent era: knowledge of own serostatus, knowledge of partner serostatus; serosorting (matching by status), and condomless anal intercourse. Serosorting overall appeared to remain common, especially with casual and one-time partners. Although PrEP use did not impact status discussion, it did impact serosorting and the likelihood of having condomless anal intercourse. For respondents not diagnosed with HIV and not on PrEP, condomless anal intercourse occurred in just over half of relationships with HIV-positive partners who were not on treatment. Biomedical prevention has intertwined with rather than supplanted seroadaptive behaviors, while contexts involving neither persist.

RESUMEN: Los comportamientos seroadaptivos ayudan a reducir el riesgo de VIH en algunos hombres que tienen sexo con otros hombres (HSH) y han sido bien documentados en varias comunidades de HSH. Los avances en prevención biomédica han cambiado los contextos de los comportamientos seroadaptivos. Por ello buscamos estimar y comparar la prevalencia de cuatro fases de la 'cascada seroadaptiva' mediante el uso de PrEP en la era reciente: conocimiento del seroestatus personal, conocimiento del seroestatus del compañero, serosorting (emparejamiento por estatus) y coito anal sin condón. En general, el serosorting parece seguir siendo común especialmente con parejas casuales o de una noche. A pesar de que el uso de PrEP no impactó la discusión sobre el estatus, sí impactó el serosorting y la probabilidad de coito anal sin condón. Los encuestados no diagnosticados con VIH y sin PrEP tuvieron coito anal sin condón en la mitad de las relaciones con parejas VIH-positivo que no estaban bajo tratamiento. La prevención biomédica se ha entremezclado en lugar de suplantar los comportamientos seropositivos, mientras persisten los contextos en los que no aparece ninguno.

Five-Step Enantioselective Synthesis of Islatravir via Asymmetric Ketone Alkynylation and an Ozonolysis Cascade.

A 5-step enantioselective synthesis of the potent anti-HIV nucleoside islatravir is reported. The highly efficient route was enabled by a novel enantioselective alkynylation of an α,ß-unsaturated ketone, a unique ozonolysis-dealkylation cascade in water, and an enzymatic aldol-glycosylation cascade.

Potent, non-covalent reversible BTK inhibitors with 8-amino-imidazo[1,5-a]pyrazine core featuring 3-position bicyclic ring substitutes.

Bruton's tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition, and for the treatment of B cell related diseases. Many BTK inhibitors have been discovered for the treatment of cancer and rheumatoid arthritis, including a series of BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine we recently reported. The X-ray crystal structures of BTK with inhibitors were also published, which provided great help for the SAR design. Here we report our SAR work introducing ring constraints for the 3-position piperidine amides on the BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine. This modification improved the potency in BTK inhibitions, as well as the PK profile and the off-target selectivity. The dose-dependent efficacy of two BTK inhibitors was observed in the rat collagen induced arthritis (CIA) model.

Prevalence of Non-medical Amphetamine Use Among Men with Diagnosed HIV Infection Who Have Sex with Men in the United States, 2015-2016.

Amphetamine use is higher among men who have sex with men (MSM) compared with other men, and is associated with sexual behavior linked to HIV transmission. No national estimates of amphetamine use among MSM with HIV have been published. We used data from the Medical Monitoring Project, a nationally representative sample of persons with diagnosed HIV, to describe patterns in amphetamine use in the past 12 months among MSM during 2015-2016 (N = 3796). Prevalence of amphetamine use in this population was 9.6% (95% CI 7.6, 11.6%) in the past 12 months. MSM who used amphetamines were more likely to have condomless sex with partners without HIV or of unknown serostatus (PR 1.87; 95% CI 1.62, 2.16) and less likely to be durably virally suppressed (PR 0.81; 95% CI 0.71, 0.91). Interventions to address amphetamine use and associated transmission risk behaviors among MSM living with HIV may decrease transmission.

Ultrahigh performance liquid chromatography methods facilitate the development of glucose-responsive insulin therapeutics.

Insulin oligosaccharide conjugates hold promise as potential glucose-responsive insulins (GRIs), which can improve the therapeutic index of insulins and mitigate the risk of hypoglycemia. A key challenge for the analytical development of such molecules is finding an efficient method to characterize the purity and impurities of conjugated insulins. Using the S-Matrix Fusion QbD-ultrahigh performance liquid chromatography (UHPLC) integrated system, we were able to quickly screen and develop two short UHPLC methods. These methods were used to support process development, clinical batch drug substance (DS) release, and stability studies of MK-2640, an insulin oligosaccharide conjugate. Both methods used a Waters CSH C18 column, with a shallow gradient of acetonitrile to aqueous mobile phase containing 25 mM sodium perchlorate and 0.05% perchloric acid. The 10-min run time method was well suited for process development and monitoring as it was able to separate the main product, MK-2640, six oligosaccharide-substituted recombinant human insulin (RHI) impurities, A21 deamidated MK-2640, and the starting material RHI. The 13-min run time method provided improved separation of the major impurities and demonstrated good chromatographic reproducibility on different instruments or using columns from different lots of stationary phase, which made it ideal for the final DS release. Validation of the 13-min method demonstrated great linearity for both the MK-2640 main peak and its related impurities, low limit of detection (0.02%), and limit of quantitation (0.05%). The high specificity of the method allowed the separation of the degradation products from main peak, thus makes it suitable for stability monitoring. The major impurities in the DS were characterized by two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS).

NHC-Catalyzed Deamination of Primary Sulfonamides: A Platform for Late-Stage Functionalization.

Herein we describe the development and application of a method for the mild, late-stage conversion of primary sulfonamides to several other other functional groups. These reactions occur via initial reductive deamination of sulfonamides to sulfinates via an NHC-catalyzed reaction of transiently formed N-sulfonylimines. The method described here is tolerant of nearly all common functional groups, as exemplified by the late-stage derivatization of several complex pharmaceutical compounds. On the basis of the prevalence of sulfonamide-containing drugs and building blocks, we have developed a method to enable sulfonamides to be applied as versatile synthetic handles for synthetic chemsitry.

Reductive Cleavage of Secondary Sulfonamides: Converting Terminal Functional Groups into Versatile Synthetic Handles.

Sulfonamides are pervasive in pharmaceuticals and agrochemicals, yet they are typically considered as terminal functional groups rather than synthetic handles. To enable the general late-stage functionalization of secondary sulfonamides, we have developed a mild and general method to reductively cleave the N-S bonds of sulfonamides to generate sulfinates and amines, components which can further react in situ to access a variety of other medicinally relevant functional groups. The utility of this platform is highlighted by the selective manipulation of several complex bioactive molecules.

Addressing Gaps in HIV Preexposure Prophylaxis Care to Reduce Racial Disparities in HIV Incidence in the United States.

The potential for human immunodeficiency virus (HIV) preexposure prophylaxis (PrEP) to reduce the racial disparities in HIV incidence in the United States might be limited by racial gaps in PrEP care. We used a network-based mathematical model of HIV transmission for younger black and white men who have sex with men (BMSM and WMSM) in the Atlanta, Georgia, area to evaluate how race-stratified transitions through the PrEP care continuum from initiation to adherence and retention could affect HIV incidence overall and disparities in incidence between races, using current empirical estimates of BMSM continuum parameters. Relative to a no-PrEP scenario, implementing PrEP according to observed BMSM parameters was projected to yield a 23% decline in HIV incidence (hazard ratio = 0.77) among BMSM at year 10. The racial disparity in incidence in this observed scenario was 4.95 per 100 person-years at risk (PYAR), a 19% decline from the 6.08 per 100 PYAR disparity in the no-PrEP scenario. If BMSM parameters were increased to WMSM values, incidence would decline by 47% (hazard ratio = 0.53), with an associated disparity of 3.30 per 100 PYAR (a 46% decline in the disparity). PrEP could simultaneously lower HIV incidence overall and reduce racial disparities despite current gaps in PrEP care. Interventions addressing these gaps will be needed to substantially decrease disparities.

Synthesis of Complex Phenols Enabled by a Rationally Designed Hydroxide Surrogate.

The conversion of aryl halides to phenols under mild reaction conditions is a longstanding and formidable challenge in organic chemistry. Herein, we report the rational design of a broadly applicable Pd-catalyzed method to prepare phenols with benzaldehyde oxime as a hydroxide surrogate. These reactions occur under mildly basic conditions and enable the late-stage hydroxylation of several functionally-dense drug-like aryl halides.

Examination of thermal unfolding and aggregation profiles of a series of developable therapeutic monoclonal antibodies.

Screening for pharmaceutically viable stability from measurements of thermally induced protein unfolding and short-term accelerated stress underpins much molecule design, selection, and formulation in the pharmaceutical biotechnology industry. However, the interrelationships among intrinsic protein conformational stability, thermal denaturation, and pharmaceutical stability are complex. There are few publications in which predictions from thermal unfolding-based screening methods are examined together with pharmaceutically relevant long-term storage stability performance. We have studied eight developable therapeutic IgG molecules under solution conditions optimized for large-scale commercial production and delivery. Thermal unfolding profiles were characterized by differential scanning calorimetry (DSC) and intrinsic fluorescence recorded simultaneously with static light scattering (SLS). These molecules exhibit a variety of thermal unfolding profiles under common reference buffer conditions and under individually optimized formulation conditions. Aggregation profiles by SE-HPLC and bioactivity upon long-term storage at 5, 25, and 40 °C establish that IgG molecules possessing a relatively wide range of conformational stabilities and thermal unfolding profiles can be formulated to achieve pharmaceutically stable drug products. Our data suggest that a formulation design strategy that increases the thermal unfolding temperature of the Fab transition may be a better general approach to improving pharmaceutical storage stability than one focused on increasing Tonset or Tm of the first unfolding transition.

Protein-silicone oil interactions: comparative effect of nonionic surfactants on the interfacial behavior of a fusion protein.

PURPOSE: To study the effect of three nonionic surfactants on the protein-silicone oil interactions. METHODS: The adsorption of Tween® 80, Pluronic® F68 and Tween® 20 at the silicone oil/water interface (using shifts in frequency (ΔF) and resistance (ΔR) with quartz crystal microbalance) was compared to the adsorption at air/water interface (using surface tension). Effect of surfactants on protein adsorption to the silicone oil/water interface was studied in sequential- and co-adsorption modes. Protein-surfactant binding in the bulk was measured using dynamic surface tension method. RESULTS: Saturation of air/water and silicone oil/water interfaces by surfactants was observed at similar bulk concentrations. ΔF due to protein adsorption to the interface decreased only when surfactant was present as a pre-adsorbed species. Insignificant differences in the dynamic surface tension values of surfactant solutions were observed in the presence of protein. CONCLUSIONS: Similar hydrophobic forces were responsible for driving the surfactant adsorption at both air/water and silicone oil/water interfaces. Surfactants were effective in reducing the protein adsorption to the silicone oil only when introduced before or along with the protein. No significant binding between the protein and surfactants was observed in the bulk.

The Relationship between Health Literacy and COVID-19 Vaccination Prevalence during a Rapidly Evolving Pandemic and Infodemic.

The gap between how health information is communicated and what people understand and can use to make informed health decisions is called health literacy. This gap was exacerbated by the rapidly changing and excessive volume of information, misinformation, and disinformation during the COVID-19 pandemic. People with lower health literacy may not have understood the importance of COVID-19 vaccination for themselves or for their communities. Our aim was to understand health literacy levels within Fulton County, Georgia, and their relationship to vaccine prevalence. Fulton county residents ages 18 and over (n = 425) completed an on-line Health Literacy Questionnaire. Individual, organizational, functional, interactive, and critical health literacy scales were created. Vaccination prevalence data were collected from the Georgia Vaccine Distribution Dashboard. All data were divided into one of three county areas. There were statistically significant variations in vaccine prevalence χ2(3) = 29.325, p < 0.001 among the three county areas. All levels of health literacy predicted overall county vaccination prevalence F (4,420) = 85.941, p < 0.001, There were significant differences in health literacy levels among two of the three county area pairs; the lowest resourced county area had the lowest vaccination prevalence and health literacy rates. This is the first example of relating direct health literacy measures across a major metropolitan US county with vaccine prevalence data.

The application of modern reactions in large-scale synthesis.

In the past decade, the field of organic synthesis has witnessed tremendous advancements in the areas of photoredox catalysis, electrochemistry, C-H activation, reductive coupling and flow chemistry. While these methods and technologies offer many strategic advantages in streamlining syntheses, their application on the process scale is complicated by several factors. In this Review, we discuss the challenges that arise when these reaction classes and/or flow chemistry technology are taken from a research laboratory operating at the milligram scale to a reactor capable of producing kilograms of product. We discuss how these challenges have been overcome through chemical and engineering solutions. Specifically, this Review will highlight key examples that have led to the production of multi-hundred-gram to kilogram quantities of active pharmaceutical ingredients or their intermediates and will provide insight on the scaling-up process to those developing new technologies and reactions.

Engineered Ribosyl-1-Kinase Enables Concise Synthesis of Molnupiravir, an Antiviral for COVID-19.

Molnupiravir (MK-4482) is an investigational antiviral agent that is under development for the treatment of COVID-19. Given the potential high demand and urgency for this compound, it was critical to develop a short and sustainable synthesis from simple raw materials that would minimize the time needed to manufacture and supply molnupiravir. The route reported here is enabled through the invention of a novel biocatalytic cascade featuring an engineered ribosyl-1-kinase and uridine phosphorylase. These engineered enzymes were deployed with a pyruvate-oxidase-enabled phosphate recycling strategy. Compared to the initial route, this synthesis of molnupiravir is 70% shorter and approximately 7-fold higher yielding. Looking forward, the biocatalytic approach to molnupiravir outlined here is anticipated to have broad applications for streamlining the synthesis of nucleosides in general.

Electronic and other new media technology interventions for HIV care and prevention: a systematic review.

INTRODUCTION: Electronic and other new media technologies (eHealth) can facilitate large-scale dissemination of information and effective delivery of interventions for HIV care and prevention. There is a need to both monitor a rapidly changing pipeline of technology-based care and prevention methods and to assess whether the interventions are appropriately diversified. We systematically review and critically appraise the research pipeline of eHealth interventions for HIV care and prevention, including published studies and other funded projects. METHODS: Two peer-reviewed literature databases were searched for studies describing the development, trial testing or implementation of new technology interventions, published from September 2014 to September 2018. The National Institutes of Health database of grants was searched for interventions still in development. Interventions were included if eHealth was utilized and an outcome directly related to HIV treatment or prevention was targeted. We summarized each intervention including the stage of development, eHealth mode of delivery, target population and stage of the HIV care and prevention continua targeted. RESULTS AND DISCUSSION: Of 2178 articles in the published literature, 113 were included with 84 unique interventions described. The interventions utilize a variety of eHealth technologies and target various points on the prevention and care continua, with greater emphasis on education, behaviour change and testing than linkage to medical care. There were a variety of interventions for HIV care support but none for PrEP care. Most interventions were developed for populations in high income countries. An additional 62 interventions with funding were found in the development pipeline, with greater emphasis on managing HIV and PrEP care. CONCLUSIONS: Our systematic review found a robust collection of eHealth interventions in the published literature as well as unpublished interventions still in development. In the published literature, there is an imbalance of interventions favouring education and behaviour change over linkage to care, retention in care, and adherence, especially for PrEP. The next generation of interventions already in the pipeline might address these neglected areas of care and prevention, but the development process is slow. Researchers need new methods for more efficient and expedited intervention development so that current and future needs are addressed.

Synthesis of Islatravir Enabled by a Catalytic, Enantioselective Alkynylation of a Ketone.

The synthesis of the potent anti-HIV investigational treatment islatravir is described. The key step in this synthesis is a highly enantioselective catalytic asymmetric alkynylation of a ketone. This reaction is a rare example of the asymmetric addition of an alkyne nucleophile to a ketone through ligand-accelerated catalysis that was performed on a greater than 100 g scale. By leveraging a multienzyme cascade, a highly diastereoselective aldol-glycosylation was used to complete the target in eight steps.

A general procedure for the preparation of beta-ketophosphonates.

A mild, high-yielding procedure for the preparation of beta-ketophosphonates is described. The condensation is general with respect to the ester and phosphonate, and the products are obtained in high yields within minutes at 0 degrees C. The reaction procedure is operationally simple and amenable to large-scale preparations.