FcγRIIA-specific DARPins as novel tools in blood cell analysis and platelet aggregation.

Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII. To generate high-affinity binders specific for FcγRIIA, libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to its extracellular domains by ribosomal display. Counterselection against FcγRIIB eliminated binders cross-reacting with both isoforms. The identified DARPins bound FcγRIIA with no detectable binding for FcγRIIB. Their affinities for FcγRIIA were in the low nanomolar range and could be enhanced by cleavage of the His-tag and dimerization. Interestingly, complex formation between DARPin and FcγRIIA followed a two-state reaction model, and discrimination from FcγRIIB was based on a single amino acid residue. In flow cytometry, DARPin F11 detected FcγRIIA+ cells even when they made up less than 1% of the cell population. Image stream analysis of primary human blood cells confirmed that F11 caused dim but reliable cell surface staining of a small subpopulation of T lymphocytes. When incubated with platelets, F11 inhibited their aggregation equally efficient as antibodies unable to discriminate between both FcγRII isoforms. The selected DARPins are unique novel tools for platelet aggregation studies as well as the role of FcγRIIA for the latent HIV-1 reservoir.

Detectability of HIV Residual Viremia despite Therapy Is Highly Associated with Treatment with a Protease Inhibitor-Based Combination Antiretroviral Therapy.

HIV persistence despite therapy contributes to chronic immune activation and inflammation, increasing the risk of aging-associated events in HIV-infected individuals. We sought here to better understand the complex link between clinical and treatment features and HIV persistence despite therapy. A total of 11,045 samples from 1,160 individuals under combination antiretroviral therapy (cART) with an unquantifiable viral load (VL; limit of quantification, 20 copies/ml) were categorized as detectable or undetectable depending on the detection of a PCR signal using a commercially available assay. Generalized estimating equation (GEE) regression was used to model viral load detectability and to assess the determinants of residual viremia (RV; VL detected below 20 copies/ml) despite therapy. A high VL zenith was associated with a higher probability to have a detectable viremia under cART. Conversely, the probability to have a detectable viral load below 20 copies/ml decreased with time under therapy. Of therapy regimens, protease inhibitor (PI)-based cART was associated with a significantly higher probability of detectable RV compared to nonnucleoside transcriptase inhibitor- or integrase inhibitor-based cART. We found that a PI-based treatment regimen is highly associated with an increased frequency of RV, supporting previous evidence suggesting that PI-based cART regimens could favor ongoing viral replication in some individuals.

On the generation of the MSD-Ñ° class of defective HIV proviruses.

Antiretroviral therapy (ART) can effectively suppress ongoing HIV replication and block disease progression, but the infection is never cured due to the persistence of a small pool of latently infected cells hosting integrated replication-competent HIV proviruses. However, the vast majority of HIV proviruses in ART-treated patients are replication-incompetent due to a variety of genetic defects. Most defective proviruses (around 90%) contain large internal deletions or are G-to-A hypermutated, resulting in destruction of most if not all viral open reading frames, which is consistent with the idea that cytotoxic T cells (CTLs) effectively remove cells that produce viral antigens. An intriguing subclass of defective proviruses (around 10%) that are consistently detected in such patients carry a small deletion or a point mutation in a relatively precise and well conserved region near the 5' end of the HIV genome, in the area that encodes the major splice donor (MSD) site and the packaging signal Ñ° in the viral RNA genome. Why this subclass of proviruses is defective has never been properly understood. We now propose a mechanistic scenario for how these MSD-Ñ° mutations can prevent viral protein expression. Based on ample results in literature, we argue that MSD inactivation triggers the activity of the 5'-polyadenylation site, resulting in the production of ultra-short non-protein-coding HIV transcripts.

The HIV-1 Tat Protein Enhances Splicing at the Major Splice Donor Site.

Transcription of the HIV-1 proviral DNA and subsequent processing of the primary transcript results in the production of a large set of unspliced and differentially spliced viral RNAs. The major splice donor site (5'ss) that is located in the untranslated leader of the HIV-1 transcript is used for the production of all spliced RNAs, and splicing at this site has to be tightly regulated to allow the balanced production of all viral RNAs and proteins. We demonstrate that the viral Tat protein, which is known to activate viral transcription, also stimulates splicing at the major 5'ss. As for the transcription effect, Tat requires the viral long terminal repeat promoter and the trans-acting responsive RNA hairpin for splicing regulation. These results indicate that HIV-1 transcription and splicing are tightly coupled processes through the coordinated action of the essential Tat protein.IMPORTANCE The HIV-1 proviral DNA encodes a single RNA transcript that is used as RNA genome and packaged into newly assembled virus particles. This full-length RNA is also used as mRNA for the production of structural and enzymatic proteins. Production of other essential viral proteins depends on alternative splicing of the primary transcript, which yields a large set of differentially spliced mRNAs. Optimal virus replication requires a balanced production of all viral RNAs, which means that the splicing process has to be strictly regulated. We show that the HIV-1 Tat protein, a factor that is well known for its transcription activating function, also stimulates splicing. Thus, Tat controls not only the level of the viral RNA but also the balance between spliced and unspliced RNAs.

What do we measure when we measure cell-associated HIV RNA.

Cell-associated (CA) HIV RNA has received much attention in recent years as a surrogate measure of the efficiency of HIV latency reversion and because it may provide an estimate of the viral reservoir size. This review provides an update on some recent insights in the biology and clinical utility of this biomarker. We discuss a number of important considerations to be taken into account when interpreting CA HIV RNA measurements, as well as different methods to measure this biomarker.

Inhibition of ROMK blocks macula densa tubuloglomerular feedback yet causes renal vasoconstriction in anesthetized rats.

The Na+-K+-2Cl- cotransporter (NKCC2) on the loop of Henle is the site of action of furosemide. Because outer medullary potassium channel (ROMK) inhibitors prevent reabsorption by NKCC2, we tested the hypothesis that ROMK inhibition with a novel selective ROMK inhibitor (compound C) blocks tubuloglomerular feedback (TGF) and reduces vascular resistance. Loop perfusion of either ROMK inhibitor or furosemide caused dose-dependent blunting of TGF, but the response to furosemide was 10-fold more sensitive (IC50 = 10-6 M for furosemide and IC50 = 10-5 M for compound C). During systemic infusion, both diuretics inhibited TGF, but ROMK inhibitor was 10-fold more sensitive (compound C: 63% inhibition; furosemide: 32% inhibition). Despite blockade of TGF, 1 h of constant systemic infusion of both diuretics reduced the glomerular filtration rate (GFR) and renal blood flow (RBF) by 40-60% and increased renal vascular resistance (RVR) by 100-200%. Neither diuretic altered blood pressure or hematocrit. Proximal tubule hydrostatic pressures (PPT) increased transiently with both diuretics (compound C: 56% increase; furosemide: 70% increase) but returned to baseline. ROMK inhibitor caused more natriuresis (3,400 vs. 1,600% increase) and calciuresis (1,200 vs. 800% increase) but less kaliuresis (33 vs. 167% increase) than furosemide. In conclusion, blockade of ROMK or Na+-K+-2Cl- transport inhibits TGF yet increases renal vascular resistance. The renal vasoconstriction was independent of volume depletion, blood pressure, TGF, or PPT.

Minor Contribution of Chimeric Host-HIV Readthrough Transcripts to the Level of HIV Cell-Associated gag RNA.

Cell-associated HIV unspliced RNA is an important marker of the viral reservoir. HIV gag RNA-specific assays are frequently used to monitor reservoir activation. Because HIV preferentially integrates into actively transcribed genes, some of the transcripts detected by these assays may not represent genuine HIV RNA but rather chimeric host-HIV readthrough transcripts. Here, we demonstrate that in HIV-infected patients on suppressive combination antiretroviral therapy, such host-derived transcripts do not significantly contribute to the HIV gag RNA level.

The design and synthesis of novel spirocyclic heterocyclic sulfone ROMK inhibitors as diuretics.

A spirocyclic class of ROMK inhibitors was developed containing a structurally diverse heterocyclic sulfone moiety and spirocyclic core starting from lead 1. These compounds not only displayed exquisite ROMK potency but significantly improved selectivity over hERG. The lead compounds were found to have favorable pharmacokinetic properties and displayed robust diuretic, natriuretic and blood pressure lowering effects in spontaneously hypertensive rats.

Improvement of hERG-ROMK index of spirocyclic ROMK inhibitors through scaffold optimization and incorporation of novel pharmacophores.

SAR in the previously described spirocyclic ROMK inhibitor series was further evolved from lead 4 by modification of the spirocyclic core and identification of novel right-side pharmacophores. In this process, it was discovered that the spiropyrrolidinone core with the carbonyl group α to the spirocenter was preferred for potent ROMK activity. Efforts aimed at decreasing hERG affinity within the series led to the discovery of multiple novel right-hand pharmacophores including 3-methoxythiadiazole, 2-methoxypyrimidine, and pyridazinone. The most promising candidate is pyridazinone analog 32 that showed an improved functional hERG/ROMK potency ratio and preclinical PK profile. In vivo evaluation of 32 demonstrated blood pressure lowering effects in the spontaneously hypertensive rat model.

The Renal Outer Medullary Potassium Channel Inhibitor, MK-7145, Lowers Blood Pressure, and Manifests Features of Bartter's Syndrome Type II Phenotype.

The renal outer medullary potassium (ROMK) channel, located at the apical surface of epithelial cells in the thick ascending loop of Henle and cortical collecting duct, contributes to salt reabsorption and potassium secretion, and represents a target for the development of new mechanism of action diuretics. This idea is supported by the phenotype of antenatal Bartter's syndrome type II associated with loss-of-function mutations in the human ROMK channel, as well as, by cardiovascular studies of heterozygous carriers of channel mutations associated with type II Bartter's syndrome. Although the pharmacology of ROMK channels is still being developed, channel inhibitors have been identified and shown to cause natriuresis and diuresis, in the absence of any significant kaliuresis, on acute oral dosing to rats or dogs. Improvements in potency and selectivity have led to the discovery of MK-7145 [5,5'-((1R,1'R)-piperazine-1,4-diylbis(1-hydroxyethane-2,1-diyl))bis(4-methylisobenzofuran-1(3H)-one)], a potential clinical development candidate. In spontaneously hypertensive rats, oral dosing of MK-7145 causes dose-dependent lowering of blood pressure that is maintained during the entire treatment period, and that displays additive/synergistic effects when administered in combination with hydrochlorothiazide or candesartan, respectively. Acute or chronic oral administration of MK-7145 to normotensive dogs led to dose-dependent diuresis and natriuresis, without any significant urinary potassium losses or changes in plasma electrolyte levels. Elevations in bicarbonate and aldosterone were found after 6 days of dosing. These data indicate that pharmacological inhibition of ROMK has potential as a new mechanism for the treatment of hypertension and/or congestive heart failure. In addition, Bartter's syndrome type II features are manifested on exposure to ROMK inhibitors.

A Subset of CD4/CD8 Double-Negative T Cells Expresses HIV Proteins in Patients on Antiretroviral Therapy.

UNLABELLED: A major goal in HIV eradication research is characterizing the reservoir cells that harbor HIV in the presence of antiretroviral therapy (ART), which reseed viremia after treatment is stopped. In general, it is assumed that the reservoir consists of CD4(+) T cells that express no viral proteins. However, recent findings suggest that this may be an overly simplistic view and that the cells that contribute to the reservoir may be a diverse population that includes both CD4(+) and CD4(-) cells. In this study, we directly infected resting CD4(+) T cells and used fluorescence-activated cell sorting (FACS) and fiber-optic array scanning technology (FAST) to identify and image cells expressing HIV Gag. We found that Gag expression from integrated proviruses occurred in resting cells that lacked surface CD4, likely resulting from Nef- and Env-mediated receptor internalization. We also extended our approach to detect cells expressing HIV proteins in patients suppressed on ART. We found evidence that rare Gag(+) cells persist during ART and that these cells are often negative for CD4. We propose that these double-negative α/ß T cells that express HIV protein may be a component of the long-lived reservoir. IMPORTANCE: A reservoir of infected cells persists in HIV-infected patients during antiretroviral therapy (ART) that leads to rebound of virus if treatment is stopped. In this study, we used flow cytometry and cell imaging to characterize protein expression in HIV-infected resting cells. HIV Gag protein can be directly detected in infected resting cells and occurs with simultaneous loss of CD4, consistent with the expression of additional viral proteins, such as Env and Nef. Gag(+) CD4(-) cells can also be detected in suppressed patients, suggesting that a subset of infected cells express proteins during ART. Understanding the regulation of viral protein expression during ART will be key to designing effective strategies to eradicate HIV reservoirs.

Differentiation of ROMK potency from hERG potency in the phenacetyl piperazine series through heterocycle incorporation.

Following the discovery of small molecule acyl piperazine ROMK inhibitors and their initial preclinical validation as a novel diuretic agent, our group set out to discover new ROMK inhibitors with reduced risk for QT effects, suitable for further pharmacological experiments in additional species. Several strategies for decreasing hERG affinity while maintaining ROMK inhibition were investigated and are described herein. The most promising candidate, derived from the newly discovered 4-N-heteroaryl acetyl series, improved functional hERG/ROMK ratio by >10× over the previous lead. In vivo evaluation demonstrated comparable diuretic effects in rat with no detectable QT effects at the doses evaluated in an in vivo dog model.

Discovery of a potent and selective ROMK inhibitor with improved pharmacokinetic properties based on an octahydropyrazino[2,1-c][1,4]oxazine scaffold.

Following the discovery of small molecule acyl piperazine ROMK inhibitors, the acyl octahydropyrazino[2,1-c][1,4]oxazine series was identified. This series displays improved ROMK/hERG selectivity, and as a consequence, the resulting ROMK inhibitors do not evoke QTc prolongation in an in vivo cardiovascular dog model. Further efforts in this series led to the discovery of analogs with improved pharmacokinetic profiles. This new series also retained comparable ROMK potency compared to earlier leads.

SAR exploration at the C-3 position of tetrahydro-ß-carboline sstr3 antagonists.

MK-4256, a tetrahydro-ß-carboline sstr3 antagonist, was discontinued due to a cardiovascular (CV) adverse effect observed in dogs. Additional investigations revealed that the CV liability (QTc prolongation) was caused by the hERG off-target activity of MK-4256 and was not due to sstr3 antagonism. In this Letter, we describe our extensive SAR effort at the C3 position of the tetrahydro-ß-carboline structure. This effort resulted in identification of 5-fluoro-pyridin-2-yl as the optimal substituent on the imidazole ring to balance sstr3 activity and the hERG off-target liability.

Pharmacologic inhibition of the renal outer medullary potassium channel causes diuresis and natriuresis in the absence of kaliuresis.

The renal outer medullary potassium (ROMK) channel, which is located at the apical membrane of epithelial cells lining the thick ascending loop of Henle and cortical collecting duct, plays an important role in kidney physiology by regulating salt reabsorption. Loss-of-function mutations in the human ROMK channel are associated with antenatal type II Bartter's syndrome, an autosomal recessive life-threatening salt-wasting disorder with mild hypokalemia. Similar observations have been reported from studies with ROMK knockout mice and rats. It is noteworthy that heterozygous carriers of Kir1.1 mutations associated with antenatal Bartter's syndrome have reduced blood pressure and a decreased risk of developing hypertension by age 60. Although selective ROMK inhibitors would be expected to represent a new class of diuretics, this hypothesis has not been pharmacologically tested. Compound A [5-(2-(4-(2-(4-(1H-tetrazol-1-yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)-one)], a potent ROMK inhibitor with appropriate selectivity and characteristics for in vivo testing, has been identified. Compound A accesses the channel through the cytoplasmic side and binds to residues lining the pore within the transmembrane region below the selectivity filter. In normotensive rats and dogs, short-term oral administration of compound A caused concentration-dependent diuresis and natriuresis that were comparable to hydrochlorothiazide. Unlike hydrochlorothiazide, however, compound A did not cause any significant urinary potassium losses or changes in plasma electrolyte levels. These data indicate that pharmacologic inhibition of ROMK has the potential for affording diuretic/natriuretic efficacy similar to that of clinically used diuretics but without the dose-limiting hypokalemia associated with the use of loop and thiazide-like diuretics.

Persistent HIV-1 transcription during ART: time to reassess its significance?

PURPOSE OF REVIEW: Despite suppressive antiretroviral therapy (ART), HIV-1 reservoirs persist and reignite viral replication if therapy is interrupted. Persistence of the viral reservoir in people with HIV-1 (PWH) is the main obstacle to an HIV-1 cure. The reservoirs are not transcriptionally silent, and viral transcripts can be detected in most ART-treated individuals. Here, we review the recent progress in the characterization of persistent HIV-1 transcription during ART. RECENT FINDINGS: Evidence from several studies indicates that, although cell-associated unspliced (US) HIV-1 RNA is abundantly expressed in ART-treated PWH, intact full-length US transcripts are rare and most US RNA is derived from defective proviruses. The transcription- and translation-competent defective proviruses, previously considered irrelevant, are increasingly being linked to residual HIV-1 pathogenesis under suppressive ART. Recent data suggest a continuous crosstalk between the residual HIV-1 activity under ART and the immune system. Persistent HIV-1 transcription on ART, despite being mostly derived from defective proviruses, predicts viral rebound upon therapy interruption, suggesting its role as an indicator of the strength of the host antiviral immune response that is shaping the viral rebound. SUMMARY: In light of the recent findings, the significance of persistent HIV-1 transcription during ART for the long-term health of PWH and the cure research should be reassessed.

Live cell screening to identify RNA-binding small molecule inhibitors of the pre-let-7-Lin28 RNA-protein interaction.

Dysregulation of the networking of RNA-binding proteins (RBPs) and RNAs drives many human diseases, including cancers, and the targeting of RNA-protein interactions (RPIs) has emerged as an exciting area of RNA-targeted drug discovery. Accordingly, methods that enable the discovery of cell-active small molecule modulators of RPIs are needed to propel this emerging field forward. Herein, we describe the application of live-cell assay technology, RNA interaction with protein-mediated complementation assay (RiPCA), for high-throughput screening to identify small molecule inhibitors of the pre-let-7d-Lin28A RPI. Utilizing a combination of RNA-biased small molecules and virtual screening hits, we discovered an RNA-binding small molecule that can disrupt the pre-let-7-Lin28 interaction demonstrating the potential of RiPCA for advancing RPI-targeted drug discovery.

Structure Guided Discovery of Novel Pan Metallo-ß-Lactamase Inhibitors with Improved Gram-Negative Bacterial Cell Penetration.

The use of ß-lactam (BL) and ß-lactamase inhibitor combination to overcome BL antibiotic resistance has been validated through clinically approved drug products. However, unmet medical needs still exist for the treatment of infections caused by Gram-negative (GN) bacteria expressing metallo-ß-lactamases. Previously, we reported our effort to discover pan inhibitors of three main families in this class: IMP, VIM, and NDM. Herein, we describe our work to improve the GN coverage spectrum in combination with imipenem and relebactam. This was achieved through structure- and property-based optimization to tackle the GN cell penetration and efflux challenges. A significant discovery was made that inhibition of both VIM alleles, VIM-1 and VIM-2, is essential for broad GN coverage, especially against VIM-producing P. aeruginosa. In addition, pharmacokinetics and nonclinical safety profiles were investigated for select compounds. Key findings from this drug discovery campaign laid the foundation for further lead optimization toward identification of preclinical candidates.

Cell-associated HIV RNA: a dynamic biomarker of viral persistence.

In most HIV-infected individuals adherent to modern antiretroviral therapy (ART), plasma viremia stays undetectable by clinical assays and therefore, additional virological markers for monitoring and predicting therapy responses and for measuring the degree of HIV persistence in patients on ART should be identified. For the above purposes, quantitation of cell-associated HIV biomarkers could provide a useful alternative to measurements of viral RNA in plasma. This review concentrates on cell-associated (CA) HIV RNA with the emphasis on its use as a virological biomarker. We discuss the significance of CA HIV RNA as a prognostic marker of disease progression in untreated patients and as an indicator of residual virus replication and the size of the dynamic viral reservoir in ART-treated patients. Potential value of this biomarker for monitoring the response to ART and to novel HIV eradication therapies is highlighted.

Translational HIV-1 research: from routine diagnostics to new virology insights in Amsterdam, the Netherlands during 1983-2013.

An HIV-1 diagnostic laboratory was established in the Academic Medical Center (AMC) of the University of Amsterdam after the discovery of human immunodeficiency virus (HIV) as the cause of the acquired immunodeficiency syndrome (AIDS). The first AIDS patients were diagnosed here in 1981 and since 1983 we have tested the samples of 50992 patients using a variety of assays that greatly improved over the years. We will describe some of the basic results from this diagnostic laboratory and then focus on the spin-off in terms of the development of novel virus assays to detect super-infections and ultra-sensitive assays to measure the intracellular HIV-1 RNA load. We also review several original research findings in the field of HIV-1 virology that stem from initial observations made in the diagnostic unit. This includes the study of genetic defects in the HIV-1 genome and time trends of the replication fitness over 30 years of viral evolution, but also the description of novel HIV-1 variants in difficult-to-diagnose clinical specimen.