Design and Synthesis of New GS-6207 Subtypes for Targeting HIV-1 Capsid Protein.

HIV-1 capsid protein (CA) is the molecular target of the recently FDA-approved long acting injectable (LAI) drug lenacapavir (GS-6207). The quick emergence of CA mutations resistant to GS-6207 necessitates the design and synthesis of novel sub-chemotypes. We have conducted the structure-based design of two new sub-chemotypes combining the scaffold of GS-6207 and the N-terminal cap of PF74 analogs, the other important CA-targeting chemotype. The design was validated via induced-fit molecular docking. More importantly, we have worked out a general synthetic route to allow the modular synthesis of novel GS-6207 subtypes. Significantly, the desired stereochemistry of the skeleton C2 was confirmed via an X-ray crystal structure of the key synthetic intermediate 22a. Although the newly synthesized analogs did not show significant potency, our efforts herein will facilitate the future design and synthesis of novel subtypes with improved potency.

Avoiding Drug Resistance in HIV Reverse Transcriptase.

HIV reverse transcriptase (RT) is an enzyme that plays a major role in the replication cycle of HIV and has been a key target of anti-HIV drug development efforts. Because of the high genetic diversity of the virus, mutations in RT can impart resistance to various RT inhibitors. As the prevalence of drug resistance mutations is on the rise, it is necessary to design strategies that will lead to drugs less susceptible to resistance. Here we provide an in-depth review of HIV reverse transcriptase, current RT inhibitors, novel RT inhibitors, and mechanisms of drug resistance. We also present novel strategies that can be useful to overcome RT's ability to escape therapies through drug resistance. While resistance may not be completely avoidable, designing drugs based on the strategies and principles discussed in this review could decrease the prevalence of drug resistance.

Specific mutations in the HIV-1 G-tract of the 3'-polypurine tract cause resistance to integrase strand transfer inhibitors.

BACKGROUND: In vitro selection experiments identified viruses resistant to integrase strand transfer inhibitors (INSTIs) carrying mutations in the G-tract (six guanosines) of the 3'-polypurine tract (3'-PPT). A clinical study also reported that mutations in the 3'-PPT were observed in a patient receiving dolutegravir monotherapy. However, recombinant viruses with the 3'-PPT mutations that were found in the clinical study were recently shown to be susceptible to INSTIs. OBJECTIVES: To identify the specific mutation(s) in the G-tract of the 3'-PPT for acquiring INSTI resistance, we constructed infectious clones bearing single or multiple mutations and systematically characterized the susceptibility of these clones to both first- and second-generation INSTIs. METHODS: The infectious clones were tested for their infectivity and susceptibility to INSTIs in a single-cycle assay using TZM-bl cells. RESULTS: A single mutation of thymidine (T) at the fifth position (GGG GTG) in the G-tract of the 3'-PPT had no effect on INSTI resistance. A double mutation, cytidine (C) or 'T' at the second position and 'T' at the fifth position (GCG GTG and GTG GTG), increased resistance to INSTIs, with the appearance of a plateau in the maximal percentage inhibition (MPI) of the dose-response curves, consistent with a non-competitive mechanism of inhibition. CONCLUSIONS: Mutations at the second and fifth positions in the G-tract of the 3'-PPT may result in complex resistance mechanism(s), rather than simply affecting INSTI binding at the IN active site.

Structure-based virtual screening workflow to identify antivirals targeting HIV-1 capsid.

We have identified novel HIV-1 capsid inhibitors targeting the PF74 binding site. Acting as the building block of the HIV-1 capsid core, the HIV-1 capsid protein plays an important role in the viral life cycle and is an attractive target for antiviral development. A structure-based virtual screening workflow for hit identification was employed, which includes docking 1.6 million commercially-available drug-like compounds from the ZINC database to the capsid dimer, followed by applying two absolute binding free energy (ABFE) filters on the 500 top-ranked molecules from docking. The first employs the Binding Energy Distribution Analysis Method (BEDAM) in implicit solvent. The top-ranked compounds are then refined using the Double Decoupling method in explicit solvent. Both docking and BEDAM refinement were carried out on the IBM World Community Grid as part of the FightAIDS@Home project. Using this virtual screening workflow, we identified 24 molecules with calculated binding free energies between - 6 and - 12 kcal/mol. We performed thermal shift assays on these molecules to examine their potential effects on the stability of HIV-1 capsid hexamer and found that two compounds, ZINC520357473 and ZINC4119064 increased the melting point of the latter by 14.8 °C and 33 °C, respectively. These results support the conclusion that the two ZINC compounds are primary hits targeting the capsid dimer interface. Our simulations also suggest that the two hit molecules may bind at the capsid dimer interface by occupying a new sub-pocket that has not been exploited by existing CA inhibitors. The possible causes for why other top-scored compounds suggested by ABFE filters failed to show measurable activity are discussed.

Rotten to the core: antivirals targeting the HIV-1 capsid core.

The capsid core of HIV-1 is a large macromolecular assembly that surrounds the viral genome and is an essential component of the infectious virus. In addition to its multiple roles throughout the viral life cycle, the capsid interacts with multiple host factors. Owing to its indispensable nature, the HIV-1 capsid has been the target of numerous antiretrovirals, though most capsid-targeting molecules have not had clinical success until recently. Lenacapavir, a long-acting drug that targets the HIV-1 capsid, is currently undergoing phase 2/3 clinical trials, making it the most successful capsid inhibitor to-date. In this review, we detail the role of the HIV-1 capsid protein in the virus life cycle, categorize antiviral compounds based on their targeting of five sites within the HIV-1 capsid, and discuss their molecular interactions and mechanisms of action. The diverse range of inhibition mechanisms provides insight into possible new strategies for designing novel HIV-1 drugs and furthers our understanding of HIV-1 biology.

Modelling changes in anxiety and depression during low-intensity cognitive behavioural therapy: An application of growth mixture models.

OBJECTIVES: Research largely supports the clinical effectiveness of low-intensity cognitive behavioural therapy (LICBT) for mild-to-moderate anxiety and depression, delivered by psychological well-being practitioners (PWPs). Knowledge regarding the predictors of treatment response, however, is relatively limited. The primary aim of this study was to model the heterogeneity in longitudinal changes in anxiety and depression throughout LICBT provided by PWPs in Northern Ireland (NI), and to explore associations between pre-treatment variables and differences in treatment response. METHODS: Growth mixture modelling (GMM) techniques were employed to examine changes in psychological status in clients (N = 253) over the first six sessions of treatment, to identify divergent early response trajectories. A series of pre-treatment variables were used to predict class membership using chi-square tests and binary logistic regression models. RESULTS: There was one class representing improvement and one representing no improvement for both anxiety and depression. Class membership was predictive of treatment outcome. Pre-treatment variables associated with less improvement included unemployment, risk of suicide, neglect of self or others, using medication, receiving previous or concurrent treatments, a longer duration of difficulties, and comorbidities. CONCLUSIONS: Findings indicate most of the sample populated an 'improvers' class for both depression and anxiety. Pre-treatment variables identified as predictive of poor treatment response may need to be considered by practitioners in potential triage referral decision policies, supporting cost-effective and efficient services. Further research around predictors of clinical outcome is recommended. PRACTITIONER POINTS: Most of the sample belonged to an 'improvers' class. Several pre-treatment variables predicted poor treatment response (unemployment, suicide risk, neglect, medication, previous or concurrent treatments, longer duration of difficulties, and comorbidities). Few studies have utilized GMM to determine predictors of outcome following LICBT Regarding pre-treatment variables, the possibility of self-report bias cannot be excluded. The time period was relatively short, although represented the optimum number of sessions recommended for LICBT. The lack of a control group and random allocation were the main limitations.

Structural basis of HIV inhibition by translocation-defective RT inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA).

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is the most potent nucleoside analog inhibitor of HIV reverse transcriptase (RT). It retains a 3'-OH yet acts as a chain-terminating agent by diminishing translocation from the pretranslocation nucleotide-binding site (N site) to the posttranslocation primer-binding site (P site). Also, facile misincorporation of EFdA-monophosphate (MP) results in difficult-to-extend mismatched primers. To understand the high potency and unusual inhibition mechanism of EFdA, we solved RT crystal structures (resolutions from 2.4 to 2.9 Å) that include inhibition intermediates (i) before inhibitor incorporation (catalytic complex, RT/DNA/EFdA-triphosphate), (ii) after incorporation of EFdA-MP followed by dT-MP (RT/DNAEFdA-MP(P)• dT-MP(N) ), or (iii) after incorporation of two EFdA-MPs (RT/DNAEFdA-MP(P)• EFdA-MP(N) ); (iv) the latter was also solved with EFdA-MP mismatched at the N site (RT/DNAEFdA-MP(P)• EFdA-MP(*N) ). We report that the inhibition mechanism and potency of EFdA stem from interactions of its 4'-ethynyl at a previously unexploited conserved hydrophobic pocket in the polymerase active site. The high resolution of the catalytic complex structure revealed a network of ordered water molecules at the polymerase active site that stabilize enzyme interactions with nucleotide and DNA substrates. Finally, decreased translocation results from favorable interactions of primer-terminating EFdA-MP at the pretranslocation site and unfavorable posttranslocation interactions that lead to observed localized primer distortions.

Preliminary Findings of a New Primary and Community Care Psychological Service in Northern Ireland: Low-Intensity Cognitive Behavioural Therapy for Common Mental Health Difficulties.

BACKGROUND: The prevalence of mental health difficulties in Northern Ireland (NI) is significantly higher than in England. In recent years, there have been extensive consultations, and subsequent recommendations made in NI in an effort to address this. AIMS: The current study aims to evaluate the effectiveness of an 'Improving Access to Psychological Therapies' (IAPT) stepped care service model using low-intensity cognitive behavioural therapy (LI-CBT) in primary and community care settings. METHOD: A pilot intervention trial design utilized two standardized outcome measures (PHQ-9 and GAD-7) before treatment (at baseline), during treatment (in every session) and at discharge (at final session). RESULTS: Preliminary reliable change outcomes for the pilot cohorts showed a recovery rate of 47.9%, improvement rate of 76.7% and deterioration rate of 6%. CONCLUSIONS: These findings suggest that the IAPT service model is clinically effective in the NI population. Data collection for the larger study was completed in December 2017. Future analyses will include follow-up data collected at 4 months post-treatment, and will also aim to identify individual and service level factors that potentially impact treatment effectiveness.

Impact of HIV-1 Integrase L74F and V75I Mutations in a Clinical Isolate on Resistance to Second-Generation Integrase Strand Transfer Inhibitors.

A novel HIV-1 integrase mutation pattern, L74F V75I, which conferred resistance to first-generation integrase strand transfer inhibitors (INSTIs), was identified in a clinical case with virological failure under a raltegravir-based regimen. Addition of L74F V75I to N155H or G140S Q148H increased resistance levels to the second-generation INSTIs dolutegravir (>385- and 100-fold, respectively) and cabotegravir (153- and 197-fold, respectively). These findings are important for the development of an accurate system for interpretation of INSTI resistance and the rational design of next-generation INSTIs.

3-Hydroxypyrimidine-2,4-Diones as Novel Hepatitis B Virus Antivirals Targeting the Viral Ribonuclease H.

Hepatitis B virus (HBV) RNase H (RNH) is an appealing therapeutic target due to its essential role in viral replication. RNH inhibitors (RNHIs) could help to more effectively control HBV infections. Here, we report 3-hydroxypyrimidine-2,4-diones as novel HBV RNHIs with antiviral activity. We synthesized and tested 52 analogs and found 4 that inhibit HBV RNH activity in infected cells. Importantly, 2 of these compounds inhibited HBV replication in the low micromolar range.

A 2-Hydroxyisoquinoline-1,3-Dione Active-Site RNase H Inhibitor Binds in Multiple Modes to HIV-1 Reverse Transcriptase.

The RNase H (RNH) function of HIV-1 reverse transcriptase (RT) plays an essential part in the viral life cycle. We report the characterization of YLC2-155, a 2-hydroxyisoquinoline-1,3-dione (HID)-based active-site RNH inhibitor. YLC2-155 inhibits both polymerase (50% inhibitory concentration [IC50] = 2.6 µM) and RNH functions (IC50 = 0.65 µM) of RT but is more effective against RNH. X-ray crystallography, nuclear magnetic resonance (NMR) analysis, and molecular modeling were used to show that YLC2-155 binds at the RNH-active site in multiple conformations.

The prevalence of non-cardiac chest pain (NCCP) using emergency department (ED) data: a Northern Ireland based study.

BACKGROUND: The aim of this study was to assess the frequency of chest pain presentations and the subsequent non-cardiac chest pain diagnoses in an emergency department (ED) over a 3 year period. METHODS: Administrative data on ED attendances to an urban general hospital in Northern Ireland between March 2013 and March 2016 were used. Data were coded and analysed to estimate frequencies of 'chest pain' presentation and the subsequent diagnoses for each year. RESULTS: Both chest pain presentations and chest pain presentations with a subsequent diagnosis of unknown cause increased each year. In total, 58.7% of all chest presentations across 3 years resulted in a non-cardiac diagnosis of either 'anxiety', 'panic' or 'chest pain of unknown cause'. DISCUSSION: There is a significant amount of patients in the ED leaving with a non-cardiac diagnosis, following an initial presentation with chest pain. CONCLUSION: Given the link between non-cardiac chest pain and frequent use of services, the degree of repeat attendance should be investigated.

Structural basis of clade-specific HIV-1 neutralization by humanized anti-V3 monoclonal antibody KD-247.

Humanized monoclonal antibody KD-247 targets the Gly(312)-Pro(313)-Gly(314)-Arg(315) arch of the third hypervariable (V3) loop of the HIV-1 surface glycoprotein. It potently neutralizes many HIV-1 clade B isolates, but not of other clades. To understand the molecular basis of this specificity, we solved a high-resolution (1.55 Å) crystal structure of the KD-247 antigen binding fragment and examined the potential interactions with various V3 loop targets. Unlike most antibodies, KD-247 appears to interact with its target primarily through light chain residues. Several of these interactions involve Arg(315) of the V3 loop. To evaluate the role of light chain residues in the recognition of the V3 loop, we generated 20 variants of KD-247 single-chain variable fragments with mutations in the antigen-binding site. Purified proteins were assessed for V3 loop binding using AlphaScreen technology and for HIV-1 neutralization. Our data revealed that recognition of the clade-specificity defining residue Arg(315) of the V3 loop is based on a network of interactions that involve Tyr(L32), Tyr(L92), and Asn(L27d) that directly interact with Arg(315), thus elucidating the molecular interactions of KD-247 with its V3 loop target.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) inhibits HIV-1 reverse transcriptase with multiple mechanisms.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a nucleoside analog that, unlike approved anti-human immunodeficiency virus type 1 (HIV-1) nucleoside reverse transcriptase inhibitors, has a 3'-OH and exhibits remarkable potency against wild-type and drug-resistant HIVs. EFdA triphosphate (EFdA-TP) is unique among nucleoside reverse transcriptase inhibitors because it inhibits HIV-1 reverse transcriptase (RT) with multiple mechanisms. (a) EFdA-TP can block RT as a translocation-defective RT inhibitor that dramatically slows DNA synthesis, acting as a de facto immediate chain terminator. Although non-translocated EFdA-MP-terminated primers can be unblocked, they can be efficiently converted back to the EFdA-MP-terminated form. (b) EFdA-TP can function as a delayed chain terminator, allowing incorporation of an additional dNTP before blocking DNA synthesis. In such cases, EFdA-MP-terminated primers are protected from excision. (c) EFdA-MP can be efficiently misincorporated by RT, leading to mismatched primers that are extremely hard to extend and are also protected from excision. The context of template sequence defines the relative contribution of each mechanism and affects the affinity of EFdA-MP for potential incorporation sites, explaining in part the lack of antagonism between EFdA and tenofovir. Changes in the type of nucleotide before EFdA-MP incorporation can alter its mechanism of inhibition from delayed chain terminator to immediate chain terminator. The versatility of EFdA in inhibiting HIV replication by multiple mechanisms may explain why resistance to EFdA is more difficult to emerge.

Hours per Patient Day: Not the Problem, Nor the Solution.

Hours per patient day (HPPD) is a metric that is easy to use in determining budgeted FTE and in comparing staffing across organizations. There are many considerations in determining the appropriate HPPD. The combination of automated patient acuity, staffing, and human resource systems provide a wealth of information for determining the budgeted HPPD and in making defensible requests for adjustments in HPPD. No matter how much data we have about staffing levels, nurse education and skill levels, the environment of care, or patient acuity, the real key is determining the outcomes we need to compare staffing against. We must quantify the savings associated with positive outcomes and get this information in the hands of the public so they can make informed decisions.

SAMHD1 has differential impact on the efficacies of HIV nucleoside reverse transcriptase inhibitors.

Sterile alpha motif- and histidine/aspartic acid domain-containing protein 1 (SAMHD1) limits HIV-1 replication by hydrolyzing deoxynucleoside triphosphates (dNTPs) necessary for reverse transcription. Nucleoside reverse transcriptase inhibitors (NRTIs) are components of anti-HIV therapies. We report here that SAMHD1 cleaves NRTI triphosphates (TPs) at significantly lower rates than dNTPs and that SAMHD1 depletion from monocytic cells affects the susceptibility of HIV-1 infections to NRTIs in complex ways that depend not only on the relative changes in dNTP and NRTI-TP concentrations but also on the NRTI activation pathways.

Biochemical, inhibition and inhibitor resistance studies of xenotropic murine leukemia virus-related virus reverse transcriptase.

We report key mechanistic differences between the reverse transcriptases (RT) of human immunodeficiency virus type-1 (HIV-1) and of xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus that can infect human cells. Steady and pre-steady state kinetics demonstrated that XMRV RT is significantly less efficient in DNA synthesis and in unblocking chain-terminated primers. Surface plasmon resonance experiments showed that the gammaretroviral enzyme has a remarkably higher dissociation rate (k(off)) from DNA, which also results in lower processivity than HIV-1 RT. Transient kinetics of mismatch incorporation revealed that XMRV RT has higher fidelity than HIV-1 RT. We identified RNA aptamers that potently inhibit XMRV, but not HIV-1 RT. XMRV RT is highly susceptible to some nucleoside RT inhibitors, including Translocation Deficient RT inhibitors, but not to non-nucleoside RT inhibitors. We demonstrated that XMRV RT mutants K103R and Q190M, which are equivalent to HIV-1 mutants that are resistant to tenofovir (K65R) and AZT (Q151M), are also resistant to the respective drugs, suggesting that XMRV can acquire resistance to these compounds through the decreased incorporation mechanism reported in HIV-1.

Building staffing science: two milestones.

Health care and nurse staffing present interesting challenges for nursing and health care leaders. The recently released Evidence and Excellence in Staffing (2nd edition) creates a framework for research and organizational improvement that leads to the development and sharing of best practices. A new model for staffing excellence has emerged with five core concepts. This new position paper calls for generating and disseminating 25-30 new best practices in nurse staffing across the care continuum over the next 3 years.

Identification of clickable HIV-1 capsid-targeting probes for viral replication inhibition.

Of the targets for HIV-1 therapeutics, the capsid core is a relatively unexploited but alluring drug target due to its indispensable roles throughout virus replication. Because of this, we aimed to identify "clickable" covalent modifiers of the HIV-1 capsid protein (CA) for future functionalization. We screened a library of fluorosulfate compounds that can undergo sulfur(VI) fluoride exchange (SuFEx) reactions, and five compounds were identified as hits. These molecules were further characterized for antiviral effects. Several compounds impacted in vitro capsid assembly. One compound, BBS-103, covalently bound CA via a SuFEx reaction to Tyr145 and had antiviral activity in cell-based assays by perturbing virus production, but not uncoating. The covalent binding of compounds that target the HIV-1 capsid could aid in the future design of antiretroviral drugs or chemical probes that will help study aspects of HIV-1 replication.

HIV-1 reverse transcriptase (RT) polymorphism 172K suppresses the effect of clinically relevant drug resistance mutations to both nucleoside and non-nucleoside RT inhibitors.

Polymorphisms have poorly understood effects on drug susceptibility and may affect the outcome of HIV treatment. We have discovered that an HIV-1 reverse transcriptase (RT) polymorphism (RT(172K)) is present in clinical samples and in widely used laboratory strains (BH10), and it profoundly affects HIV-1 susceptibility to both nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) when combined with certain mutations. Polymorphism 172K significantly suppressed zidovudine resistance caused by excision (e.g. thymidine-associated mutations) and not by discrimination mechanism mutations (e.g. Q151M complex). Moreover, it attenuated resistance to nevirapine or efavirenz imparted by NNRTI mutations. Although 172K favored RT-DNA binding at an excisable pre-translocation conformation, it decreased excision by thymidine-associated mutation-containing RT. 172K affected DNA handling and decreased RT processivity without significantly affecting the k(cat)/K(m) values for dNTP. Surface plasmon resonance experiments revealed that RT(172K) decreased DNA binding by increasing the dissociation rate. Hence, the increased zidovudine susceptibility of RT(172K) results from its increased dissociation from the chain-terminated DNA and reduced primer unblocking. We solved a high resolution (2.15 Å) crystal structure of RT mutated at 172 and compared crystal structures of RT(172R) and RT(172K) bound to NNRTIs or DNA/dNTP. Our structural analyses highlight differences in the interactions between α-helix E (where 172 resides) and the active site ß9-strand that involve the YMDD loop and the NNRTI binding pocket. Such changes may increase dissociation of DNA, thus suppressing excision-based NRTI resistance and also offset the effect of NNRTI resistance mutations thereby restoring NNRTI binding.