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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.

Advances in adoptive transfer immunotherapy have found beneficial effects for Interleukin-15 (IL-15) in the graft versus tumor (GvT) activity of NK and cytotoxic T cells. However, the benefits of this activity must be weighed against the increased risk of acute graft versus host disease (GvHD) in patients. We propose that BNZ-2, a peptide antagonist of IL-15 and IL-21 signaling, as a therapeutic option for GvHD that may relieve intestinal pathologies while retaining adoptive transfer activity. As a novel model of intestinal inflammatory disease, we have found that humanization with peripheral blood mononuclear cells (PBMCs) of NOG mice with transgenic expression of human IL-15 (NOG-IL15) consistently catalyzes the onset of intestinal GvHD within twenty days of engraftment. To investigate whether BNZ-2 can inhibit IL-15-catalized intestinal GvHD we treated NOG-IL15 mice and assessed the degree of intestinal GvHD and the immunophenotype of engrafted cells following twenty days of humanization with four million unmanipulated PBMCs. BNZ-2 treatment inhibited with equal efficacy to anti-IL-15 the localization of immune cells to the intestinal lamina propria protecting the tissue from inflammation-induced loss of tissue integrity. However, BNZ-2 treatment retained the systemic engraftment of the blood and spleen, including NK and CD8 cytotoxic T-cells. These data support BNZ-2 as a therapeutic candidate for GvHD treatment, describe a new model of intestinal GvHD, and suggest that a therapeutic window exists that separates intestinal GvHD pathologies from the benefits of adoptive cell transfer. Citation Format: Kevin R. Kipp, Nick Doerr, Laith Q. Al-Mawsawi, Woo Jae Kim, Adrian J. Giovannone, Nazli Azimi. BNZ-2, a dual specific IL15/IL21 inhibitor, rescues humanized NOG-IL15 transgenic mice from intestinal acute graft versus host disease without disrupting NK and CD8 T cell engraftment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB143.

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.

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.

Background: The γc-family of cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, IL-21) control major immune responses and lymphocyte development. However, aberrant γc cytokine activity contributes to or pathologically drives human diseases including T-cell malignancies, graft-versus-host disease, and numerous autoimmune diseases (e.g., alopecia areata), where IL-2, IL-9, and IL-15 have been specifically implicated. Importantly, these multi-cytokine diseases are not effectively and safely treated by the currently available anti-cytokine approaches. Methods: BNZ-1 is the first of a novel class of rationally-designed, multi-cytokine inhibitors that selectively inhibits IL-2 and IL-15, and to a lesser degree, IL-9 signaling through the γc receptor, without affecting IL-4, IL-7, or IL-21. Preclinical proof-of-concept has been demonstrated in several animal models of T-cell malignancies (Nata et al., 2015 J Biol Chem), including Large Granular Lymphocyte Leukemia (LGL) and Adult T-cell Leukemia. In this open-label, single-dose, dose-escalation, first-in-human study conducted at a single center in the United States (NCT03046459), 18 healthy adults (n=3/cohort) received a single 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 safety and sample collection for 30 days. Results: All subjects completed the study. BNZ-1 was considered well-tolerated with a good safety profile with no serious or severe adverse events (AEs), no dose-limiting toxicities, and no clinically-significant changes on clinical labs (serum biochemistry, hematology, liver enzymes), vital signs or electrocardiograms. Headache was the only AE reported in ≥1 subject (n=3). BNZ-1 exposure was generally dose proportional with an elimination half-life of ~5 days across the range of doses tested, which is consistent with other PEGylated peptides and supports weekly or every other week dosing. The pharmacodynamic activity of BNZ-1 was characterized by flow cytometry of PBMCs obtained on Days 4, 15 and 31 and used to calculate the maximum change from baseline (Emax) for regulatory T-cells (Tregs; IL-2 effect), NK cells (IL-15 effect) and CD8+ central memory T-cells (Tcm; IL-2 & IL-15 effect). For Tregs, a ~50-60% decrease was observed on Day 4 after doses of 0.4 to 1.6mg/kg, while doses of 3.2 and 6.4mg/kg produced an 80-93% decrease from baseline that peaked on Day 15. NK cells were decreased from baseline on Day 4 by 20%, 40% and 60% at 0.2, 0.4 and 0.8mg/kg, respectively, and plateaued at 70 to 80% decrease at doses ≥1.6 mg/kg. Tcm were decreased at Day 4 for the three highest dose cohorts that continued to decline to Day 15 when all doses, except 0.2 mg/kg, showed a mean decrease ranging from 10 to 81% that generally trended with dose. Tregs, NK cells, and Tcm returned to/toward baseline by 30 days post dose. Post-dose counts of CD4+ and CD8+ T-cells, B-cells, and monocytes were unchanged at all time points studied. Conclusions: These preliminary clinical data suggest that BNZ-1 is a highly-active, selective immunomodulator that safely decreases Tregs, NK cells and Tcm, while leaving the major leukocyte populations unaffected. These data support the planned Phase 1/2 dose-ranging study in LGL and Cutaneous T-cell Lymphoma. Disclosures Frohna: Bioniz Therapeutics: Employment. Tagaya: Bioniz Therapeutics: Equity Ownership. Ratnayake: Bioniz Therapeutics: Consultancy. Doerr: Bioniz Therapeutics: Employment. Basheer: Bioniz Therapeutics: Employment. Al-Mawsawi: Bioniz Therapeutics: Employment. Kim: Bioniz Therapeutics: Employment. Azimi: Bioniz Therapeutics: Employment.

HIV-1 integrase (IN) is an essential enzyme for viral infection. Here, we report an extensive π electron orbital interaction between four amino acids, W132, M178, F181 and F185, located at the dimeric interface of IN that is critical for the strand transfer activity alone. Catalysis of nine different mutant IN proteins at these positions were evaluated. Whereas the 3′-processing activity is predominantly strong, the strand transfer activity of each enzyme was completely dependent on an intact π electron orbital interaction at the dimeric interface. Four representative IN mutants were constructed in the context of the infectious NL4.3 HIV-1 viral clone. Whereas viruses with an intact π electron orbital interaction at the IN dimeric interface replicated comparable to wild type, viruses containing an abolished π interaction were non-infectious. Q-PCR analysis of viral DNA forms during viral replication revealed pleiotropic effects of most mutations. We hypothesize that the π interaction is a critical contact point for the assembly of functional IN multimeric complexes, and that IN multimerization is required for a functional pre-integration complex. The rational design of small molecule inhibitors targeting the disruption of this π–π interaction should lead to powerful anti-retroviral drugs.

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.

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.

The β-diketoacid class of HIV-1 integrase (IN) inhibitors represent the first potent class of compounds specific for the strand transfer catalytic activity of the viral enzyme. Previously, utilizing a β-diketoacid pharmacophore as a search query, we identified a substituted 2-pyrrolinone with modest IN inhibitory activity from a database of small-molecules [Dayam, R.; Sanchez, T.; Neamati, N. J. Med. Chem. 2005, 48, 8009]. In efforts to optimize this class of IN inhibitors, we carried out a structure–activity relationship analysis around the 2-pyrrolinone core. Here, we present a new class of 2-pyrrolinone IN inhibitors.

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.

Recently, we reported small-molecule chalcones as a novel class of HIV-1 integrase (IN) inhibitors. The most potent compound showed an IC50 value of 2 microM for both IN-mediated 3'-processing and strand transfer reactions. To further utilize the chalcones, we developed pharmacophore models to identify chemical signatures important for biological activity. The derived models were validated with a collection of published inhibitors, and then were applied to screen a subset of our small molecule database. We tested 71 compounds in an in vitro assay specific for IN enzymatic activity. Forty-four compounds showed inhibitory potency100 microM, and four of them exhibited IC50 values10 microM. One compound, 62, with an IC50 value of 0.6 microM, displayed better potency than the original chalcone 2 against the strand transfer process. This study demonstrates the systematic use of pharmacophore technologies to discover novel structurally diverse inhibitors based on lead molecules that would exhibit poor characteristics in vivo. The identified compounds have the potential to exhibit favorable pharmacokinetic and pharmacodynamic profiles.

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.

The human epidermal growth factor receptor (HER) family is a highly explored and promising anticancer drug target. At present, several investigational agents targeted to the HER family of receptors are in various stages of development. Five drugs are already in the clinic for the treatment of cancers that overexpress HER family receptors. Two FDA-approved small-molecule drugs, gefitinib and erlotinib, inhibit HER1 tyrosine kinase activity. Two mAbs, cetuximab and panitumumab, target the extracellular domain of HER1, and another, trastuzumab, targets the extracellular domain of HER2. HER2 is a prominent member of the HER family of receptor tyrosine kinases and serves as a preferred dimerization partner for other HER family members. This paper reviews recently patented small-molecule inhibitors of HER2 receptor kinase activity, and inhibitors of HER2 expression and shedding. Apart from the well-explored quinazoline class of compounds (e.g., lapatinib), arylazole, benzodithiazole, pyrrolopyridazine, pyrrolot...

Herein, we report the identification of a unique HIV-1 integrase (IN) inhibitor-binding site using photoaffinity labeling and mass spectrometric analysis. We chemically incorporated a photo-activatable benzophenone moiety into a series of coumarin-containing IN inhibitors. A representative of this series was covalently photo-crosslinked with the IN core domain and subjected to HPLC purification. Fractions were subsequently analyzed by using MALDI-MS and electrospray ionization (ESI)-MS to identify photo-crosslinked products. In this fashion, a single binding site for an inhibitor located within the tryptic peptide 128 AACWWAGIK 136 was identified. Site-directed mutagenesis followed by in vitro inhibition assays resulted in the identification of two specific amino acid residues, C130 and W132, in which substitutions resulted in a marked resistance to the IN inhibitors. Docking studies suggested a specific disruption in functional oligomeric IN complex formation. The combined approach of photo-affinity labeling/MS analysis with site-directed mutagenesis/molecular modeling is a powerful approach for elucidating inhibitor-binding sites of proteins at the atomic level. This approach is especially important for the study of proteins that are not amenable to traditional x-ray crystallography and NMR techniques. This type of structural information can help illuminate processes of inhibitor resistance and thereby facilitate the design of more potent second-generation inhibitors.

The stator of the bacterial flagellar motor is formed from the membrane proteins MotA and MotB, which associate in complexes with stoichiometry MotA(4)MotB(2) (Kojima, S., and Blair, D. F., preceding paper in this issue). The MotA/MotB complexes conduct ions across the membrane, and couple ion flow to flagellar rotation by a mechanism that appears to involve conformational changes within the complex. MotA has four membrane-crossing segments, termed A1-A4, and MotB has one, termed B. We are studying the organization of the 18 membrane segments in the MotA(4)MotB(2) complex by using targeted disulfide cross-linking. A previous cross-linking study showed that the two B segments in the complex (one from each MotB subunit) are arranged as a symmetrical dimer of alpha-helices. Here, we extend the cross-linking study to segments A3 and A4. Single Cys residues were introduced by mutation in several consecutive positions in segments A3 and A4, and double mutants were made by pairwise combination of subsets of the Cys replacements in segments A3, A4, and B. Disulfide cross-linking of the single- and double-Cys proteins was studied in whole cells, in membranes, and in detergent solution. Several combinations of Cys residues in segments A3 and B gave a high yield of disulfide-linked MotA/MotB heterodimer upon oxidation with iodine. Positions of efficient cross-linking identify a helix face on segment A3 that is in proximity to segment(s) B. Some combinations of Cys residues in segments A4 and B also gave a significant yield of disulfide-linked heterodimer, indicating that segment A4 is also near segment(s) B. Certain combinations of Cys residues in segments A3 and A4 cross-linked to form MotA tetramers in high yield upon oxidation. The high-yield positions identify faces on A3 and A4 that are at an interface between MotA subunits. Taken together with mutational studies and patterns of amino acid conservation, the cross-linking results delineate the overall arrangement of 10 membrane segments in the MotA/MotB complex, and identify helix faces likely to line the proton channels.

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.

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.

A novel genome-wide genetics platform is presented in this study, which permits functional interrogation of all point mutations across a viral genome in parallel. Here we generated the first fitness profile of individual point mutations across the influenza virus genome. Critical residues on the viral genome were systematically identified, which provided a collection of subdomain data informative for structure-function studies and for effective rational drug and vaccine design. Our data was consistent with known, well-characterized structural features. In addition, we have achieved a validation rate of 68% for severely attenuated mutations and 94% for neutral mutations. The approach described in this study is applicable to other viral or microbial genomes where a means of genetic manipulation is available.

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.

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., Author Summary The emergence of drug resistance during antiviral treatment limits treatment options and poses challenges to pharmaceutical development. Meanwhile, the search for novel antiviral compounds with chemical genetic screens has led to the identification of antiviral agents with undefined drug mechanisms. Daclatasvir, an effective NS5A inhibitor, is one such example. In traditional methods to identify critical residues governing drug-protein interactions, wild type virus is passaged under drug treatment pressure, enabling the identification of resistant mutations evolved after multiple viral passages. However, this method only characterizes a fraction of the positively selected variants. Here we have simultaneously quantified the relative change in replication fitness as well as the relative sensitivity to Daclatasvir for all possible single amino acid mutations in the NS5A domain IA, thereby identifying the entire panel of positions that interact with the drug. Using mathematical models, we predicted which mutations pose the greatest risk of causing emergence of resistance under different scenarios of treatment compliance. The mutant fitness and drug-sensitivity profiles obtained can also inform the patient-specific use of Daclatasvir and may facilitate the development of second-generation drugs with a higher genetic barrier to resistance.

By combining the high efficiency enrichment via our continuous flow magnetic separation (CFMS) technique with the analytical power of next-generation sequencing, we have enabled the generation of antibody mimetics with a single round of mRNA display. This approach eliminates iterative selection cycles, providing a path to fully automated ligand generation to meet the goal of a renewable, proteome-wide affinity reagent resource.

HIV-1 integrase (IN) is one of three essential enzymes for viral replication, and is a focus of ardent antiretroviral drug discovery and development efforts. Diligent research has led to the development of the strand-transfer-specific chemical class of IN inhibitors, with two compounds from this group, raltegravir and elvitegravir, advancing the farthest in the US Food and Drug Administration (FDA) approval process for any IN inhibitor discovered thus far. Raltegravir, developed by Merck & Co., has been approved by the FDA for HIV-1 therapy, whereas elvitegravir, developed by Gilead Sciences and Japan Tobacco, has reached phase III clinical trials. Although this is an undoubted success for the HIV-1 IN drug discovery field, the emergence of HIV-1 IN strand-transfer-specific drug-resistant viral strains upon clinical use of these compounds is expected. Furthermore, the problem of strand-transfer-specific IN drug resistance will be exacerbated by the development of cross-resistant viral strains due to an overlapping binding orientation at the IN active site and an equivalent inhibitory mechanism for the two compounds. This inevitability will result in no available IN-targeted therapeutic options for HIV-1 treatment-experienced patients. The development of allosterically targeted IN inhibitors presents an extremely advantageous approach for the discovery of compounds effective against IN strand-transfer drug-resistant viral strains, and would likely show synergy with all available FDA-approved antiretroviral HIV-1 therapeutics, including the IN strand-transfer-specific compounds. Herein we review the concept of allosteric IN inhibition, and the small molecules that have been investigated to bind non-active-site regions to inhibit IN function.

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.

HIV-1 integrase (IN) is one of three essential enzymes for viral replication, and is a focus of ardent antiretroviral drug discovery and development efforts. Diligent research has led to the development of the strand-transfer-specific chemical class of IN inhibitors, with two compounds from this group, raltegravir and elvitegravir, advancing the farthest in the US Food and Drug Administration (FDA) approval process for any IN inhibitor discovered thus far. Raltegravir, developed by Merck & Co., has been approved by the FDA for HIV-1 therapy, whereas elvitegravir, developed by Gilead Sciences and Japan Tobacco, has reached phase III clinical trials. Although this is an undoubted success for the HIV-1 IN drug discovery field, the emergence of HIV-1 IN strand-transfer-specific drug-resistant viral strains upon clinical use of these compounds is expected. Furthermore, the problem of strand-transfer-specific IN drug resistance will be exacerbated by the development of cross-resistant viral strains due to an overlapping binding orientation at the IN active site and an equivalent inhibitory mechanism for the two compounds. This inevitability will result in no available IN-targeted therapeutic options for HIV-1 treatment-experienced patients. The development of allosterically targeted IN inhibitors presents an extremely advantageous approach for the discovery of compounds effective against IN strand-transfer drug-resistant viral strains, and would likely show synergy with all available FDA-approved antiretroviral HIV-1 therapeutics, including the IN strand-transfer-specific compounds. Herein we review the concept of allosteric IN inhibition, and the small molecules that have been investigated to bind non-active-site regions to inhibit IN function.

HIV-1 integrase (IN) catalyzes the integration of proviral DNA into the host genome, an essential step for viral replication. Inhibition of IN catalytic activity provides an attractive strategy for antiretroviral drug design. Currently two IN inhibitors, MK-0518 and GS-9137, are in advanced stages of human clinical trials. The IN inhibitors in clinical evaluation demonstrate excellent antiretroviral efficacy alone or in combination regimens as compared to previously used clinical antiretroviral agents in naive and treatment-experienced HIV-1 infected patients. However, the emergence of viral strains resistant to clinically studied IN inhibitors and the dynamic nature of the HIV-1 genome demand a continued effort toward the discovery of novel inhibitors to keep a therapeutic advantage over the virus. Continued efforts in the field have resulted in the discovery of compounds from diverse chemical classes. In this review, we provide a comprehensive report of all IN inhibitors discovered in the years 2005 and 2006. © 2007 Wiley Periodicals, Inc. Med Res Rev, 28, No. 1, 118–154, 2008

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.

HIV-1 integrase (IN) catalyzes the integration of proviral DNA into the host genome, an essential step for viral replication. Inhibition of IN catalytic activity provides an attractive strategy for antiretroviral drug design. Currently two IN inhibitors, MK-0518 and GS-9137, are in advanced stages of human clinical trials. The IN inhibitors in clinical evaluation demonstrate excellent antiretroviral efficacy alone or in combination regimens as compared to previously used clinical antiretroviral agents in naive and treatment-experienced HIV-1 infected patients. However, the emergence of viral strains resistant to clinically studied IN inhibitors and the dynamic nature of the HIV-1 genome demand a continued effort toward the discovery of novel inhibitors to keep a therapeutic advantage over the virus. Continued efforts in the field have resulted in the discovery of compounds from diverse chemical classes. In this review, we provide a comprehensive report of all IN inhibitors discovered in the years 2005 and 2006. © 2007 Wiley Periodicals, Inc. Med Res Rev, 28, No. 1, 118–154, 2008

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.

The prospect of HIV-1 integrase (IN) as a therapeutically viable retroviral drug target is on the verge of realization. The observed preclinical and clinical performance of beta-diketo containing and naphthyridine carboxamide compounds provides direct proof for the clinical application of IN inhibition. These validated lead compounds are useful in the design and development of second generation IN inhibitors. The results from preclinical and clinical studies on the first generation IN inhibitors reiterate a demand for novel second generation inhibitors with improved pharmacokinetic and metabolic properties. Pharmacophore-based drug design techniques facilitate the discovery of novel compounds on the basis of validated lead compounds specific for a drug target. In this article we have comprehensively reviewed the application of pharmacophore-based drug design methods in the field of IN inhibitor discovery.

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.

Trade-offs between throughput, read length, and error rates in high-throughput sequencing limit certain applications such as monitoring viral quasispecies. Here, we describe a molecular-based tag linkage method that allows assemblage of short sequence reads into long DNA fragments. It enables haplotype phasing with high accuracy and sensitivity to interrogate individual viral sequences in a quasispecies. This approach is demonstrated to deduce ∼ 2000 unique 1.3 kb viral sequences from HIV-1 quasispecies in vivo and after passaging ex vivo with a detection limit of ∼ 0.005% to ∼ 0.001%. Reproducibility of the method is validated quantitatively and qualitatively by a technical replicate. This approach can improve monitoring of the genetic architecture and evolution dynamics in any quasispecies population.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12977-014-0124-6) contains supplementary material, which is available to authorized users.