Antiviral Properties of HIV-1 Capsid Inhibitor GSK878.

GSK878 is a newly described HIV-1 inhibitor that binds to the mature capsid (CA) hexamer in a pocket originally identified as the binding site of the well-studied CA inhibitor PF-74. Here, we show that GSK878 is highly potent, inhibiting an HIV-1 reporter virus in MT-2 cells with a mean 50% effective concentration (EC50) of 39 pM and inhibiting a panel of 48 chimeric viruses containing diverse CA sequences with a mean EC50 of 94 pM. CA mutations associated with reduced susceptibility to other inhibitors that bind to PF-74 binding site (L56I, M66I, Q67H, N74D, T107N, and Q67H/N74D) also reduced susceptibility to GSK878, with M66I, Q67H/N74D, and L56I having the greatest impact on antiviral activity. Amino acid substitutions in the CA cyclophilin A (CypA) binding loop (H87P and P90A), distal from the inhibitor binding site and associated with reduced CA-CypA binding, subtly, but reproducibly, also decreased GSK878 potency. Mechanism-of-action studies showed that GSK878 blocked both early (preintegration) and late (postintegration) steps in HIV-1 replication, with the early inhibition primarily determining the compound's antiviral activity. The early inhibition results from blocks to HIV-1 nuclear import and proviral integration and is associated with altered stability of the HIV-1 CA core.

Scaffold modifications to the 4-(4,4-dimethylpiperidinyl) 2,6-dimethylpyridinyl class of HIV-1 allosteric integrase inhibitors.

Allosteric integrase inhibitors (ALLINIs) of HIV-1 may hold promise as a novel mechanism for HIV therapeutics and cure. Scaffold modifications to the 4-(4,4-dimethylpiperidinyl) 2,6-dimethylpyridinyl class of ALLINIs provided a series of potent compounds with differentiated 5/6 fused ring systems. Notably, inhibitors containing the 1,2,4-triazolopyridine and imidazopyridine core exhibited single digit nM antiviral potency and low to moderate clearance after intravenous (IV) dosing in rat pharmacokinetic (PK) studies. The 1,2,4-triazolopyridines showed a higher oral exposure when compared to the imidazopyridines. Further modifications to the C5 substituent of the 1,2,4-triazolopyridines resulted in a new lead compound, which had improved rat IV/PO PK compared to the former lead compound GSK3739936, while maintaining antiviral potency. Structure-activity relationships (SAR) and rat pharmacokinetic profiles of this series are discussed.

Cross-Reactive Antibody Responses to Novel H5Nx Influenza Viruses Following Homologous and Heterologous Prime-Boost Vaccination with a Prepandemic Stockpiled A(H5N1) Vaccine in Humans.

Recently, novel highly pathogenic avian influenza H5Nx viruses (clade 2.3.4.4) caused outbreaks in US poultry. We evaluated the potential of a stockpiled A(H5N1) A/Anhui/1/2005 (clade 2.3.4) vaccine to elicit cross-reactive antibody responses to these emerging viruses. Sera from subjects who received 2 doses of MF59-adjuvanted A/Anhui/1/2005, or 1 dose of MF59-adjuvanted A/Anhui/1/2005 following priming with a clade 1 vaccine were characterized by microneutralization assays and modified hemagglutination inhibition (HI) assays. Only heterologous prime-boost vaccination induced modest cross-reactive HI antibody responses to H5Nx viruses. Heterologous prime-boost may provide a more effective vaccination strategy to broaden the antibody responses to emerging viruses.

Effect of Priming With Seasonal Influenza A(H3N2) Virus on the Prevalence of Cross-Reactive Hemagglutination-Inhibition Antibodies to Swine-Origin A(H3N2) Variants.

Background: Recent outbreaks of swine-origin influenza A(H3N2) variant (H3N2v) viruses have raised public health concerns. Previous studies indicated that older children and young adults had the highest levels of hemagglutination-inhibition (HI) antibodies to 2010-2011 H3N2v viruses. However, newly emerging 2013 H3N2v have acquired antigenic mutations in the hemagglutinin at amino acid position 145 (N145K/R). We estimated the levels of serologic cross-reactivity among humans primed with seasonal influenza A(H3N2) (sH3N2), using postinfection ferret antisera. We also explored age-related HI antibody responses to 2012-2013 H3N2v viruses. Methods: Human and ferret antisera were tested in HI assays against 1 representative 2012 H3N2v (145N) and 2 2013 H3N2v (145K/R) viruses, together with 9 sH3N2 viruses circulating since 1968. Results: Low levels of cross-reactivity between the H3N2v and sH3N2 viruses from the 1970s-1990s were observed using postinfection ferret antisera. The overall seroprevalence among the sH3N2-primed population against 2012-2013 H3N2v viruses was >50%, and age-related seroprevalence was observed. Seroprevalence was significantly higher to 2013 H3N2v than to 2012 H3N2v viruses among some children likely to have been primed with A/Sydney/5/97-like (145K) or A/Wuhan/359/95-like viruses (145K). Conclusions: A single substitution (N145K/R) was sufficient to affect seropositivity to H3N2v viruses in some individuals. Insight into age-related antibody responses to newly emerging H3N2v viruses is critical for risk assessment and pandemic preparedness.

Highly pathogenic Avian Influenza A(H5N1) virus infection among workers at live bird markets, Bangladesh, 2009-2010.

The risk for influenza A(H5N1) virus infection is unclear among poultry workers in countries where the virus is endemic. To assess H5N1 seroprevalence and seroconversion among workers at live bird markets (LBMs) in Bangladesh, we followed a cohort of workers from 12 LBMs with existing avian influenza surveillance. Serum samples from workers were tested for H5N1 antibodies at the end of the study or when LBM samples first had H5N1 virus-positive test results. Of 404 workers, 9 (2%) were seropositive at baseline. Of 284 workers who completed the study and were seronegative at baseline, 6 (2%) seroconverted (7 cases/100 poultry worker-years). Workers who frequently fed poultry, cleaned feces from pens, cleaned food/water containers, and did not wash hands after touching sick poultry had a 7.6 times higher risk for infection compared with workers who infrequently performed these behaviors. Despite frequent exposure to H5N1 virus, LBM workers showed evidence of only sporadic infection.

The HIV-1 Capsid-Targeted Inhibitor GSK878 Alters Selection of Target Sites for HIV DNA Integration.

Decades of effort have yielded highly effective antiviral agents to treat HIV, but viral strains have evolved resistance to each inhibitor type, focusing attention on the importance of developing new inhibitor classes. A particularly promising new target is the HIV capsid, the function of which can be disrupted by highly potent inhibitors that persist long term in treated subjects. Studies with such inhibitors have contributed to an evolving picture of the role of capsid itself-the inhibitors, like certain capsid protein (CA) amino acid substitutions, can disrupt intracellular trafficking to alter the selection of target sites for HIV DNA integration in cellular chromosomes. In this study, we compare effects on HIV integration targeting for two potent inhibitors-a new molecule targeting CA, GSK878, and the previously studied lenacapavir (LEN, formerly known as GS-6207). We find that both inhibitors reduce integration in active transcription units and near epigenetic marks associated with active transcription. A careful study of integration near repeated sequences indicated frequencies were also altered for integration within multiple repeat classes. One notable finding was increased integration in centromeric satellite repeats in the presence of LEN and GSK878, which is of interest because proviruses integrated in centromeric repeats have been associated with transcriptional repression, inducibility, and latency. These data add to the picture that CA protein remains associated with preintegration complexes through the point in infection during which target sites for integration are selected, and specify new aspects of the consequences of disrupting this mechanism.

Potent Long-Acting Inhibitors Targeting the HIV-1 Capsid Based on a Versatile Quinazolin-4-one Scaffold.

Long-acting (LA) human immunodeficiency virus-1 (HIV-1) antiretroviral therapy characterized by a ≥1 month dosing interval offers significant advantages over daily oral therapy. However, the criteria for compounds that enter clinical development are high. Exceptional potency and low plasma clearance are required to meet dose size requirements; excellent chemical stability and/or crystalline form stability is required to meet formulation requirements, and new antivirals in HIV-1 therapy need to be largely free of side effects and drug-drug interactions. In view of these challenges, the discovery that capsid inhibitors comprising a quinazolinone core tolerate a wide range of structural modifications while maintaining picomolar potency against HIV-1 infection in vitro, are assembled efficiently in a multi-component reaction, and can be isolated in a stereochemically pure form is reported herein. The detailed characterization of a prototypical compound, GSK878, is presented, including an X-ray co-crystal structure and subcutaneous and intramuscular pharmacokinetic data in rats and dogs.

Discovery and Preclinical Profiling of GSK3839919, a Potent HIV-1 Allosteric Integrase Inhibitor.

Allosteric HIV-1 integrase inhibitors (ALLINIs) have been of interest recently because of their novel mechanism of action. Strategic modifications to the C5 moiety of a class of 4-(4,4-dimethylpiperidinyl)-2,6-dimethylpyridinyl ALLINIs led to the identification of a tetrahydroisoquinoline heterocycle as a suitable spacer element to project the distal hydrophobic aryl ring. Subsequent optimization of the aryl substitutions identified 12 as an ALLINI with single-digit nanomolar inhibitory potency and low clearance across preclinical species. In preclinical toxicology studies with 12 in rats, lipid hepatocellular vacuolation was observed. Removal of the C6 methyl group resulted in GSK3839919 (22), which exhibited a reduced incidence and severity of lipid vacuolation in both in vitro assays and in vivo studies while maintaining the potency and pharmacokinetic (PK) properties of the prototype. The virology, PK, and toxicology profiles of 22 are discussed.

Genome Sequences of Microbacterium foliorum Phages Anseraureola, Pondwater, and Yasuo.

Anseraureola, Pondwater, and Yasuo are bacteriophages with siphovirus morphology that infect Microbacterium foliorum NRRL B-24224. They were isolated from soil collected in Amherst, Massachusetts, and have genome lengths between 17,362 bp and 17,453 bp. These phages each contain 25 predicted protein-coding genes and are assigned to phage cluster EE.

Racial and ethnic disparities in H1N1 vaccine uptake in Medicaid: Lessons learned for a COVID-19 vaccine rollout.

BACKGROUND: Racial and ethnic minorities in the US have been disproportionately affected by the COVID-19 pandemic and are at risk for disparities in COVID-19 vaccinations. The H1N1 flu vaccine experience provides lessons learned to address and prevent racial and ethnic disparities in COVID-19 vaccinations. We aim to identify racial/ethnic and geographic disparities in H1N1 vaccinations among Medicaid enrollees to inform equitable COVID-19 vaccination policies and strategies. METHODS: The study population included people under 65 who were continuously enrolled in Medicaid in 2009 and 2010 from 28 states and the District of Columbia. H1N1 vaccinations were identified from Medicaid outpatient claims. Vaccination rates were calculated for the overall sample and subpopulations by race/ethnicity and state. RESULTS: 3,708,894 (12.3%) Medicaid enrollees in the sample were vaccinated for H1N1 in 2009-2010. Race-specific vaccination rates ranged from 8.1% in American Indian/Alaska Native (AI/AN) to 19.8% in Asian/Pacific Islander Medicaid enrollees. NHB enrollees had lower vaccination rates than non-Hispanic White (NHW) enrollees in all states, with the exceptions of Maryland, Missouri, Ohio, and Washington. The largest disparity between NHB and NHW was in Pennsylvania (1.0% vs. 7.0%), while the largest absolute difference between NHB and NHW enrollees was in Georgia (17.4% vs. 30.7%). CONCLUSIONS: Our study found huge variation in H1N1 vaccinations across states and racial/ethnic disparities in H1N1 vaccinations within states. In most states, NHB and AI/AN Medicaid enrollees had lower vaccination rates than Whites. Hispanic and Asian/Pacific Islander Medicaid enrollees in most states had higher vaccination rates than Whites.

Discovery of BMS-986144, a Third-Generation, Pan-Genotype NS3/4A Protease Inhibitor for the Treatment of Hepatitis C Virus Infection.

The discovery of a pan-genotypic hepatitis C virus (HCV) NS3/4A protease inhibitor based on a P1-P3 macrocyclic tripeptide motif is described. The all-carbon tether linking the P1-P3 subsites of 21 is functionalized with alkyl substituents, which are shown to effectively modulate both potency and absorption, distribution, metabolism, and excretion (ADME) properties. The CF3Boc-group that caps the P3 amino moiety was discovered to be an essential contributor to metabolic stability, while positioning a methyl group at the C1 position of the P1' cyclopropyl ring enhanced plasma trough values following oral administration to rats. The C7-fluoro, C6-CD3O substitution pattern of the P2* isoquinoline heterocycle of 21 was essential to securing the targeted potency, pharmacokinetic (PK), and toxicological profiles. The C6-CD3O redirected metabolism away from a problematic pathway, thereby circumventing the time-dependent cytochrome P (CYP) 450 inhibition observed with the C6-CH3O prototype.

A general solution for unstable boronic acids: slow-release cross-coupling from air-stable MIDA boronates.

Many boronic acids, including 2-heterocyclic, vinyl, and cyclopropyl derivatives, are inherently unstable, which can limit their benchtop storage and/or efficient cross-coupling. We herein report the first general solution to this problem: in situ slow release of unstable boronic acids from the corresponding air-stable MIDA boronates. This remarkably general approach has transformed all three classes of these unstable boronic acids into shelf-stable and highly effective building blocks for cross-coupling with a wide range of aryl and heteroaryl chlorides.

Multistep synthesis of complex boronic acids from simple MIDA boronates.

Due to its sensitivity to most synthetic reagents, it is typically necessary to introduce the boronic acid functional group just prior to its utilization. Overcoming this important limitation, we herein report that air- and chromatographically stable MIDA boronates are compatible with a wide range of common reagents which enables the multistep synthesis of complex boronic acid building blocks from simple B-containing starting materials. X-ray and variable temperature NMR studies link the unique stability of MIDA boronates to a kinetic inaccessibility of the potentially reactive boron p-orbital and/or nitrogen lone pair. These findings were collectively harnessed to achieve a short and modular total synthesis of (+)-crocacin C via the iterative cross-coupling of a structurally complex, MIDA-protected haloboronic acid building block.

Applications of Fluorine in Medicinal Chemistry.

The role of fluorine in drug design and development is expanding rapidly as we learn more about the unique properties associated with this unusual element and how to deploy it with greater sophistication. The judicious introduction of fluorine into a molecule can productively influence conformation, pKa, intrinsic potency, membrane permeability, metabolic pathways, and pharmacokinetic properties. In addition, (18)F has been established as a useful positron emitting isotope for use with in vivo imaging technology that potentially has extensive application in drug discovery and development, often limited only by convenient synthetic accessibility to labeled compounds. The wide ranging applications of fluorine in drug design are providing a strong stimulus for the development of new synthetic methodologies that allow more facile access to a wide range of fluorinated compounds. In this review, we provide an update on the effects of the strategic incorporation of fluorine in drug molecules and applications in positron emission tomography.

Synthesis of many different types of organic small molecules using one automated process.

Small-molecule synthesis usually relies on procedures that are highly customized for each target. A broadly applicable automated process could greatly increase the accessibility of this class of compounds to enable investigations of their practical potential. Here we report the synthesis of 14 distinct classes of small molecules using the same fully automated process. This was achieved by strategically expanding the scope of a building block-based synthesis platform to include even C(sp3)-rich polycyclic natural product frameworks and discovering a catch-and-release chromatographic purification protocol applicable to all of the corresponding intermediates. With thousands of compatible building blocks already commercially available, many small molecules are now accessible with this platform. More broadly, these findings illuminate an actionable roadmap to a more general and automated approach for small-molecule synthesis.

Preexisting Immunity, More Than Aging, Influences Influenza Vaccine Responses.

Background. Influenza disproportionately impacts older adults while current vaccines have reduced effectiveness in the older population. Methods. We conducted a comprehensive evaluation of cellular and humoral immune responses of adults aged 50 years and older to the 2008-2009 seasonal trivalent inactivated influenza vaccine and assessed factors influencing vaccine response. Results. Vaccination increased hemagglutination inhibition and neutralizing antibody; however, 66.3% of subjects did not reach hemagglutination inhibition titers ≥ 40 for H1N1, compared with 22.5% for H3N2. Increasing age had a minor negative impact on antibody responses, whereas prevaccination titers were the best predictors of postvaccination antibody levels. Preexisting memory B cells declined with age, especially for H3N2. However, older adults still demonstrated a significant increase in antigen-specific IgG(+) and IgA(+) memory B cells postvaccination. Despite reduced frequency of preexisting memory B cells associated with advanced age, fold-rise in memory B cell frequency in subjects 60+ was comparable to subjects age 50-59. Conclusions. Older adults mounted statistically significant humoral and cell-mediated immune responses, but many failed to reach hemagglutination inhibition titers ≥40, especially for H1N1. Although age had a modest negative effect on vaccine responses, prevaccination titers were the best predictor of postvaccination antibody levels, irrespective of age.

General method for synthesis of 2-heterocyclic N-methyliminodiacetic acid boronates.

A wide range of 2-pyridyl and other difficult-to-access heterocyclic N-methyliminodiacetic acid boronates can be readily prepared from the corresponding bromides via a new method involving direct transligation of 2-heterocyclic trialkoxyborate salts with N-methyliminodiacetic acid (MIDA) at elevated temperatures.