Identification of a γc Receptor Antagonist That Prevents Reprogramming of Human Tissue-resident Cytotoxic T Cells by IL15 and IL21.

BACKGROUND & AIMS: Gamma chain (γc) cytokines (interleukin [IL]2, IL4, IL7, IL9, IL15, and IL21) signal via a common γc receptor. IL2 regulates the immune response, whereas IL21 and IL15 contribute to development of autoimmune disorders, including celiac disease. We investigated whether BNZ-2, a peptide designed to inhibit IL15 and IL21, blocks these cytokines selectively and its effects on intraepithelial cytotoxic T cells. METHODS: We obtained duodenal biopsies from 9 patients with potential celiac disease (positive results from tests for anti-TG2 but no villous atrophy), 30 patients with untreated celiac disease (with villous atrophy), and 5 patients with treated celiac disease (on a gluten-free diet), as well as 43 individuals without celiac disease (controls). We stimulated primary intestinal intraepithelial CD8+ T-cell lines, or CD8+ T cells directly isolated from intestinal biopsies, with γc cytokines in presence or absence of BNZ-2. Cells were analyzed by immunoblots, flow cytometry, or RNA-sequencing analysis for phosphorylation of signaling molecules, gene expression profiles, proliferation, and levels of granzyme B. RESULTS: Duodenal tissues from patients with untreated celiac disease had increased levels of messenger RNAs encoding IL15 receptor subunit alpha (IL15RA) and IL21 compared with tissues from patients with potential celiac disease and controls. Activation of intraepithelial cytotoxic T cells with IL15 or IL21 induced separate signaling pathways; incubation of the cells with IL15 and IL21 cooperatively increased their transcriptional activity, proliferation, and cytolytic properties. BNZ-2 specifically inhibited the effects of IL15 and IL21, but not of other γc cytokines. CONCLUSIONS: We found increased expression of IL15RA and IL21 in duodenal tissues from patients with untreated celiac disease compared with controls. IL15 and IL21 cooperatively activated intestinal intraepithelial cytotoxic T cells. In particular, they increased their transcriptional activity, proliferation, and cytolytic activity. The peptide BNZ-2 blocked these effects, but not those of other γc cytokines, including IL2. BNZ-2 might be used to prevent cytotoxic T-cell-mediated tissue damage in complex immune disorders exhibiting upregulation of IL15 and IL21.

Correction: Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality.

[This corrects the article DOI: 10.1371/journal.pgen.1005310.].

Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality.

Viruses often encode proteins with multiple functions due to their compact genomes. Existing approaches to identify functional residues largely rely on sequence conservation analysis. Inferring functional residues from sequence conservation can produce false positives, in which the conserved residues are functionally silent, or false negatives, where functional residues are not identified since they are species-specific and therefore non-conserved. Furthermore, the tedious process of constructing and analyzing individual mutations limits the number of residues that can be examined in a single study. Here, we developed a systematic approach to identify the functional residues of a viral protein by coupling experimental fitness profiling with protein stability prediction using the influenza virus polymerase PA subunit as the target protein. We identified a significant number of functional residues that were influenza type-specific and were evolutionarily non-conserved among different influenza types. Our results indicate that type-specific functional residues are prevalent and may not otherwise be identified by sequence conservation analysis alone. More importantly, this technique can be adapted to any viral (and potentially non-viral) protein where structural information is available.

A quantitative high-resolution genetic profile rapidly identifies sequence determinants of hepatitis C viral fitness and drug sensitivity.

Widely used chemical genetic screens have greatly facilitated the identification of many antiviral agents. However, the regions of interaction and inhibitory mechanisms of many therapeutic candidates have yet to be elucidated. Previous chemical screens identified Daclatasvir (BMS-790052) as a potent nonstructural protein 5A (NS5A) inhibitor for Hepatitis C virus (HCV) infection with an unclear inhibitory mechanism. Here we have developed a quantitative high-resolution genetic (qHRG) approach to systematically map the drug-protein interactions between Daclatasvir and NS5A and profile genetic barriers to Daclatasvir resistance. We implemented saturation mutagenesis in combination with next-generation sequencing technology to systematically quantify the effect of every possible amino acid substitution in the drug-targeted region (domain IA of NS5A) on replication fitness and sensitivity to Daclatasvir. This enabled determination of the residues governing drug-protein interactions. The relative fitness and drug sensitivity profiles also provide a comprehensive reference of the genetic barriers for all possible single amino acid changes during viral evolution, which we utilized to predict clinical outcomes using mathematical models. We envision that this high-resolution profiling methodology will be useful for next-generation drug development to select drugs with higher fitness costs to resistance, and also for informing the rational use of drugs based on viral variant spectra from patients.

High-throughput identification of loss-of-function mutations for anti-interferon activity in the influenza A virus NS segment.

UNLABELLED: Viral proteins often display several functions which require multiple assays to dissect their genetic basis. Here, we describe a systematic approach to screen for loss-of-function mutations that confer a fitness disadvantage under a specified growth condition. Our methodology was achieved by genetically monitoring a mutant library under two growth conditions, with and without interferon, by deep sequencing. We employed a molecular tagging technique to distinguish true mutations from sequencing error. This approach enabled us to identify mutations that were negatively selected against, in addition to those that were positively selected for. Using this technique, we identified loss-of-function mutations in the influenza A virus NS segment that were sensitive to type I interferon in a high-throughput fashion. Mechanistic characterization further showed that a single substitution, D92Y, resulted in the inability of NS to inhibit RIG-I ubiquitination. The approach described in this study can be applied under any specified condition for any virus that can be genetically manipulated. IMPORTANCE: Traditional genetics focuses on a single genotype-phenotype relationship, whereas high-throughput genetics permits phenotypic characterization of numerous mutants in parallel. High-throughput genetics often involves monitoring of a mutant library with deep sequencing. However, deep sequencing suffers from a high error rate (∼0.1 to 1%), which is usually higher than the occurrence frequency for individual point mutations within a mutant library. Therefore, only mutations that confer a fitness advantage can be identified with confidence due to an enrichment in the occurrence frequency. In contrast, it is impossible to identify deleterious mutations using most next-generation sequencing techniques. In this study, we have applied a molecular tagging technique to distinguish true mutations from sequencing errors. It enabled us to identify mutations that underwent negative selection, in addition to mutations that experienced positive selection. This study provides a proof of concept by screening for loss-of-function mutations on the influenza A virus NS segment that are involved in its anti-interferon activity.

High-throughput profiling of point mutations across the HIV-1 genome.

BACKGROUND: The HIV-1 pandemic is not the result of a static pathogen but a large genetically diverse and dynamic viral population. The virus is characterized by a highly mutable genome rendering efforts to design a universal vaccine a significant challenge and drives the emergence of drug resistant variants upon antiviral pressure. Gaining a comprehensive understanding of the mutational tolerance of each HIV-1 genomic position is therefore of critical importance. RESULTS: Here we combine high-density mutagenesis with the power of next-generation sequencing to gauge the replication capacity and therefore mutational tolerability of single point mutations across the entire HIV-1 genome. We were able to achieve the evaluation of point mutational effects on viral replicative capacity for 5,553 individual HIV-1 nucleotide positions - representing 57% of the viral genome. Replicative capacity was assessed at 3,943 nucleotide positions for a single alternate base change, 1,459 nucleotide positions for two alternate base changes, and 151 nucleotide positions for all three possible alternate base changes. This resulted in the study of how a total of 7,314 individual point mutations impact HIV-1 replication on a single experimental platform. We further utilize the dataset for a focused structural analysis on a capsid inhibitor binding pocket. CONCLUSION: The approach presented here can be applied to any pathogen that can be genetically manipulated in a laboratory setting. Furthermore, the methodology can be utilized under externally applied selection conditions, such as drug or immune pressure, to identify genetic elements that contribute to drug or host interactions, and therefore mutational routes of pathogen resistance and escape.

Systematic identification of H274Y compensatory mutations in influenza A virus neuraminidase by high-throughput screening.

Compensatory mutations contribute to the appearance of the oseltamivir resistance substitution H274Y in the neuraminidase (NA) gene of H1N1 influenza viruses. Here, we describe a high-throughput screening method utilizing error-prone PCR and next-generation sequencing to comprehensively screen NA genes for H274Y compensatory mutations. We found four mutations that can either fully (R194G, E214D) or partially (L250P, F239Y) compensate for the fitness deficiency of the H274Y mutant. The compensatory effect of E214D is applicable in both seasonal influenza virus strain A/New Caledonia/20/1999 and 2009 pandemic swine influenza virus strain A/California/04/2009. The technique described here has the potential to profile a gene at the single-nucleotide level to comprehend the dynamics of mutation space and fitness and thus offers prediction power for emerging mutant species.

Single-round, multiplexed antibody mimetic design through mRNA display.

In a single round: By combining the high-efficiency enrichment through the continuous-flow magnetic separation (CFMS) technique with the analytical power of next-generation sequencing, the generation of antibody mimetics with a single round of mRNA display is made possible. This approach eliminates iterative selection cycles and provides a path to fully automated ligand generation (see picture).

Are we living in the end of the blockbuster drug era?

For the last two decades, we have seen remarkable growth in the pharmaceutical industry. This growth has mainly been due to the approximately 100 new blockbuster drugs, such as Lipitor® (atorvastatin) and Plavix® (clopidogrel). More than half of the revenue of major pharmaceutical companies and above one-third of the total pharmaceutical revenues came from the sales of these blockbuster drugs. Questions concerning the fate of these blockbuster drugs are beginning to surface as they are approaching their patent expiration dates, and as they are expected to face significant competition from generic versions. Branded drugs with more than USD 120 billion in sales (as of 2008) are expected to lose their patent protection in the next 3 to 4 years, while the less expensive generic versions are ready to enter the market. It is plausible that a major paradigm shift in our thinking is needed to stay innovative, competitive and economically feasible in this new era of drug development. A new wave of innovations is expected to boost the blockbuster regime. Herein, we discuss the different threats facing the branded monopoly, as well as some of the hopeful expectations for the blockbuster drug.

Results From a First-in-Human Study of BNZ-1, a Selective Multicytokine Inhibitor Targeting Members of the Common Gamma (γc) Family of Cytokines.

Pathologic roles of interleukin (IL)-2, IL-9, and IL-15, have been implicated in multiple T-cell malignancies and autoimmune diseases. BNZ-1 is a selective and simultaneous inhibitor of IL-2, IL-9, and IL-15, which targets the common gamma chain signaling receptor subunit. In this first-in-human study, 18 healthy adults (n = 3/cohort) received an intravenous dose of 0.2, 0.4, 0.8, 1.6, 3.2, or 6.4 mg/kg infused over ≤5 minutes on day 1 and were followed for 30 days for safety and pharmacokinetic/pharmacodynamic sample collection. No dose-limiting toxicities, infusion reactions, or serious or severe treatment-emergent adverse events were observed. Headache was the only treatment-emergent adverse event in >1 subject (n = 3). Peak and total BNZ-1 exposure was generally dose proportional, with a terminal elimination half-life of ∼5 days. Pharmacodynamic effects of BNZ-1 on regulatory T cells (Tregs, IL-2), natural killer (NK) cells (IL-15) and CD8 central memory T cells (Tcm, IL-15) were measured by flow cytometry and used to demonstrate target engagement. For Tregs, 0.2 mg/kg was an inactive dose, while a maximum ∼50% to 60% decrease from baseline was observed on day 4 after doses of 0.4 to 1.6 mg/kg, and higher doses produced an 80% to 93% decrease from baseline on day 15. Similar pharmacodynamic trends were observed for natural killer cells and CD8 Tcm, although decreases in CD8 Tcm were more prolonged. These subpopulations returned to/toward baseline by day 31. T cells (total, CD4, and CD8), B cells, and monocytes were unchanged throughout. These preliminary results suggest that BNZ-1 safely and selectively inhibits IL-2 and IL-15, which results in robust, reversible immunomodulation.

Blocking interactions between HIV-1 integrase and cellular cofactors: an emerging anti-retroviral strategy.

HIV-1 integrase (IN) executes the insertion of proviral DNA into the host cell genome, an essential step in the retroviral life cycle. This is a multi-step process that starts in the cytosol and culminates in the nucleus of the infected cell. It is becoming increasingly clear that IN interacts with a wide range of different host-cell proteins throughout the viral life cycle. These cellular cofactors are exploited for various functions, including nuclear import, DNA target-site selection and virion assembly. The disruption of key interactions between IN and direct cellular cofactors affords a novel therapeutic approach for the design and development of new classes of anti-retroviral agents. Here, we will discuss the rationale behind this emerging and promising therapeutic strategy for HIV drug discovery. Our discussion includes the identified IN cellular cofactors, key research developments in the field and the implications this approach will have on the current HIV treatment regimen.

Inhibitory profile of a LEDGF/p75 peptide against HIV-1 integrase: insight into integrase-DNA complex formation and catalysis.

A lens epithelium-derived growth factor (LEDGF)/p75 peptide was evaluated for human immunodeficiency virus type 1 integrase (IN) inhibitory activity. The LEDGF/p75 peptide modestly inhibited IN catalysis and was dependent on IN-DNA assembly. The peptide was also effective at disrupting LEDGF/p75-IN complex formation. We next investigated the activity of the LEDGF/p75 peptide on IN mutant proteins that are unable to catalyze the DNA strand transfer reaction. The LEDGF/p75 peptide displayed an increased potency on these IN proteins, from 5-fold to greater than 10-fold, indicating the IN multimeric state greatly influences the peptide inhibitory effects. These results shed light on IN-DNA multimeric formation, and how this process influences the LEDGF/p75-IN interaction.

Quinolone 3-carboxylic acid pharmacophore: design of second generation HIV-1 integrase inhibitors.

Two decades of intensive research efforts have led to the discovery of a large number of HIV-1 integrase (IN) inhibitors. Recently, the United States Food and Drug Administration (US FDA) approved MK-0518, or raltegravir ( 1), as the first IN inhibitor for HIV/AIDS treatment. Growing clinical evidence also demonstrates that the emergence of HIV-1 virus strains bearing IN amino acid substitutions that confer resistance to IN inhibitors is inevitable. The discovery of second generation inhibitors with potency against viral strains bearing drug resistant IN substitutions is necessary for ongoing effective treatment of viral infections. We generated common feature pharmacophore hypotheses using a training set of quinolone 3-carboxylic acid IN inhibitors, including the clinical candidate GS-9137 ( 2). A database search of small molecules using the quinolone 3-carboxylic acid pharmacophore model, followed by in vitro evaluation of selected hits in an assay specific to IN, resulted in the discovery of potential leads with diverse structural scaffolds useful for the design of second generation IN inhibitors.

Anti-infectives: clinical progress of HIV-1 integrase inhibitors.

BACKGROUND: HIV-1 integrase (IN) represents a therapeutically advantageous viral target to treat HIV/AIDS in the clinic. Over a decade of progress in the field has resulted in IN inhibitor chemical classes that display specificity for strand transfer catalysis of the enzyme, thus blocking viral DNA integration into host cell nuclear DNA, an essential step for viral infectivity. OBJECTIVE: In this manuscript we provide an update on recent HIV-1 IN inhibitors that have been clinically evaluated, which include MK-0518, MK-2048, GS-9137, GS-9160, GS-9224, GSK-364735, and BMS-707035. The information presented here can aid in the IN drug developmental process. METHODS: We have limited the scope of this review to information available on the clinical evaluation of promising strand transfer-specific IN inhibitors and their potential drug-drug interaction profiles with other antiretroviral agents. RESULTS/CONCLUSION: The development of strand transfer-specific inhibitor classes is an important achievement for the IN drug design and development field. However, continued drug development is needed given that the ability of HIV to replicate under therapeutic pressure will undoubtedly lead to the emergence of IN drug-resistant viral strains.

Single amino acid substitution in HIV-1 integrase catalytic core causes a dramatic shift in inhibitor selectivity.

HIV-1 integrase (IN) mediates the insertion of viral cDNA into the cell genome, a vital process for replication. This step is catalyzed by two separate DNA reaction events, termed 3'-processing and strand transfer. Here, we show that six inhibitors from five structurally different classes of compounds display a selectivity shift towards preferential strand transfer inhibition over the 3'-processing activity of IN when a single serine is substituted at position C130. Even though IN utilizes the same active site for both reactions, this finding suggests a distinct conformational dissimilarity in the mechanistic details of each IN catalytic event.

Discovery of novel non-cytotoxic salicylhydrazide containing HIV-1 integrase inhibitors.

The previously discovered salicylhydrazide class of compounds displayed potent HIV-1 integrase (IN) inhibitory activity. The development of this class of compounds as antiretroviral agents was halted due to cytotoxicity in the nanomolar to sub-micromolar range. We identified a novel class of non-cytotoxic hydrazide IN inhibitors utilizing the minimally required salicylhydrazide substructure as a template in a small-molecule database search. The novel hydrazides displayed low micromolar IN inhibitory activity and are several hundred-fold less cytotoxic than previously disclosed salicylhydrazide IN inhibitors.

HIV-1 integrase inhibitors: an emerging clinical reality.

From the discovery of HIV-1 integrase (IN) inhibitors using enzyme-based assays in 1992, it has taken 15 years to achieve success in human clinical trials. Currently available antiretroviral drugs set high clinical standards in efficacy and long-term safety for upcoming novel HIV/AIDS therapeutic agents. The results from advanced stages of human clinical trials with IN inhibitors indicate a promising future for these compounds as a novel class of antiretroviral drugs. Success and failure of previously discovered antiretroviral drugs have taught us that there are no magic bullets in eradicating HIV. However, approval of drugs selectively targeting IN has long been awaited. There is once again a surge of interest in the field focusing on clinical development of IN inhibitors. Here, we summarise the current status of IN inhibitors under clinical development. These agents include S-1360, GSK-364735, L-870,810, L-870,812, MK-0518, GS-9137, L-900564, GS-9224, and BMS-707035. Promising antiviral activity has already been achieved with MK-0518 and GS-9137 in late-stage clinical studies.

From ligand to complexes: inhibition of human immunodeficiency virus type 1 integrase by beta-diketo acid metal complexes.

beta-Diketo acid-containing compounds are a promising class of human immunodeficiency virus type 1 (HIV-1) integrase (IN) inhibitors. Starting from the hypothesis that these inhibitors are able to coordinate ions in solution before interacting on the active site, a series of potentiometric measurements have been performed to understand the coordination ability of the diketo acid pharmacophore toward the biologically relevant Mg(2+). Moreover, by using beta-diketo acid/ester as model ligands with a set of divalent metal ions (Mg, Mn, Ni, Co, Cu, and Zn), we obtained a series of complexes and tested them for anti-HIV-1 IN activity. Results demonstrate that the diketo acid functionality chelates divalent metal ions in solution, and complexes with metals in different stoichiometric ratios are isolated. We postulate that the diketo acids act as complexes in their active form. In particular, they predominantly form species such as Mg(2)L(2+) and Mg(2)L(2) (derived from diketo acids, H(2)L), and MgL(+) and MgL(2) (derived from diketo esters, HL) at physiological pH. Furthermore, the synthesized mono- and dimetallic complexes inhibited IN at a high nanomolar to low micromolar range, with metal dependency in the phenyl diketo acid series. Retrospective analysis suggests that the electronic properties of the aromatic framework influence the metal-chelating ability of the diketo acid system. Therefore, the difference in activities is related to the complexes they preferentially form in solution, and these findings are important for the design of a new generation of IN inhibitors.

Short communication: HIV-1 gag genetic variation in a single acutely infected participant defined by high-resolution deep sequencing.

Acute HIV-1 infection is characterized by the rapid generation of highly diverse genetic variants to adapt to the new host environment. Understanding the dynamics of viral genetic variation at this stage of infection is critical for vaccine design efforts and early drug treatment. Here, using a high-resolution deep sequencing approach targeting the HIV-1 gag region, we reveal very early immune pressure with dramatic subpopulation shifts in a single acutely infected participant providing further insight into the genetic dynamics of acute HIV-1 infection.

High-throughput profiling of influenza A virus hemagglutinin gene at single-nucleotide resolution.

Genetic research on influenza virus biology has been informed in large part by nucleotide variants present in seasonal or pandemic samples, or individual mutants generated in the laboratory, leaving a substantial part of the genome uncharacterized. Here, we have developed a single-nucleotide resolution genetic approach to interrogate the fitness effect of point mutations in 98% of the amino acid positions in the influenza A virus hemagglutinin (HA) gene. Our HA fitness map provides a reference to identify indispensable regions to aid in drug and vaccine design as targeting these regions will increase the genetic barrier for the emergence of escape mutations. This study offers a new platform for studying genome dynamics, structure-function relationships, virus-host interactions, and can further rational drug and vaccine design. Our approach can also be applied to any virus that can be genetically manipulated.