Structure-based discovery of CFTR potentiators and inhibitors.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, whereas its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here, we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify CFTR modulators. We docked ∼155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered mid-nanomolar potentiators, as well as inhibitors, that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.

Structure-based discovery of CFTR potentiators and inhibitors.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, while its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify novel CFTR modulators. We docked ~155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered novel mid-nanomolar potentiators as well as inhibitors that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.

A macrocyclic peptide inhibitor traps MRP1 in a catalytically incompetent conformation.

Adenosine triphosphate-binding cassette (ABC) transporters, such as multidrug resistance protein 1 (MRP1), protect against cellular toxicity by exporting xenobiotic compounds across the plasma membrane. However, constitutive MRP1 function hinders drug delivery across the blood-brain barrier, and MRP1 overexpression in certain cancers leads to acquired multidrug resistance and chemotherapy failure. Small-molecule inhibitors have the potential to block substrate transport, but few show specificity for MRP1. Here we identify a macrocyclic peptide, named CPI1, which inhibits MRP1 with nanomolar potency but shows minimal inhibition of a related multidrug transporter P-glycoprotein. A cryoelectron microscopy (cryo-EM) structure at 3.27 Å resolution shows that CPI1 binds MRP1 at the same location as the physiological substrate leukotriene C4 (LTC4). Residues that interact with both ligands contain large, flexible sidechains that can form a variety of interactions, revealing how MRP1 recognizes multiple structurally unrelated molecules. CPI1 binding prevents the conformational changes necessary for adenosine triphosphate (ATP) hydrolysis and substrate transport, suggesting it may have potential as a therapeutic candidate.

Sex and prior exposure jointly shape innate immune responses to a live herpesvirus vaccine.

Background: Both sex and prior exposure to pathogens are known to influence responses to immune challenges, but their combined effects are not well established in humans, particularly in early innate responses critical for shaping subsequent outcomes. Methods: We employed systems immunology approaches to study responses to a replication-defective, herpes simplex virus (HSV) 2 vaccine in men and women either naive or previously exposed to HSV. Results: Blood transcriptomic and cell population profiling showed substantial changes on day 1 after vaccination, but the responses depended on sex and whether the vaccinee was naive or previously exposed to HSV. The magnitude of early transcriptional responses was greatest in HSV naive women where type I interferon (IFN) signatures were prominent and associated negatively with vaccine-induced neutralizing antibody titers, suggesting that a strong early antiviral response reduced the uptake of this replication-defective virus vaccine. While HSV seronegative vaccine recipients had upregulation of gene sets in type I IFN (IFN-α/ß) responses, HSV2 seropositive vaccine recipients tended to have responses focused more on type II IFN (IFN-γ) genes. Conclusions: These results together show that prior exposure and sex interact to shape early innate responses that then impact subsequent adaptive immune phenotypes. Funding: Intramural Research Program of the NIH, the National Institute of Allergy and Infectious Diseases, and other institutes supporting the Trans-NIH Center for Human Immunology, Autoimmunity, and Inflammation. The vaccine trial was supported through a clinical trial agreement between the National Institute of Allergy and Infectious Diseases and Sanofi Pasteur. Clinical trial number: NCT01915212.

Serum and Cervicovaginal Fluid Antibody Profiling in Herpes Simplex Virus-Seronegative Recipients of the HSV529 Vaccine.

Previous herpes simplex virus type 2 (HSV-2) vaccines have not prevented genital herpes. Concerns have been raised about the choice of antigen, the type of antibody induced by the vaccine, and whether antibody is present in the genital tract where infection occurs. We reported results of a trial of an HSV-2 replication-defective vaccine, HSV529, that induced serum neutralizing antibody responses in 78% of HSV-1-/HSV-2- vaccine recipients. Here we show that HSV-1-/HSV-2- vaccine recipients developed antibodies to epitopes of several viral proteins; however, fewer antibody epitopes were detected in vaccine recipients compared with naturally infected persons. HSV529 induced antibodies that mediated HSV-2-specific natural killer (NK) cell activation. Depletion of glycoprotein D (gD)-binding antibody from sera reduced neutralizing titers by 62% and NK cell activation by 81%. HSV-2 gD antibody was detected in cervicovaginal fluid at about one-third the level of that in serum. A vaccine that induces potent serum antibodies transported to the genital tract might reduce HSV genital infection.

A Randomized, Double-Blinded, Placebo-Controlled, Phase 1 Study of a Replication-Defective Herpes Simplex Virus (HSV) Type 2 Vaccine, HSV529, in Adults With or Without HSV Infection.

BACKGROUND: Herpes simplex virus 2 (HSV2) causes genital herpes in >400 million persons worldwide. METHODS: We conducted a randomized, double-blinded, placebo-controlled trial of a replication-defective HSV2 vaccine, HSV529. Twenty adults were enrolled in each of 3 serogroups of individuals: those negative for both HSV1 and HSV2 (HSV1-/HSV2-), those positive or negative for HSV1 and positive for HSV2 (HSV1±/HSV2+), and those positive for HSV1 and negative for HSV2 (HSV1+/HSV2-). Sixty participants received vaccine or placebo at 0, 1, and 6 months. The primary end point was the frequency of solicited local and systemic reactions to vaccination. RESULTS: Eighty-nine percent of vaccinees experienced mild-to-moderate solicited injection site reactions, compared with 47% of placebo recipients (95% confidence interval [CI], 12.9%-67.6%; P = .006). Sixty-four percent of vaccinees experienced systemic reactions, compared with 53% of placebo recipients (95% CI, -17.9% to 40.2%; P = .44). Seventy-eight percent of HSV1-/HSV2- vaccine recipients had a ≥4-fold increase in neutralizing antibody titer after 3 doses of vaccine, whereas none of the participants in the other serogroups had such responses. HSV2-specific CD4+ T-cell responses were detected in 36%, 46%, and 27% of HSV1-/HSV2-, HSV1±/HSV2+, and HSV1+/HSV2- participants, respectively, 1 month after the third dose of vaccine, and CD8+ T-cell responses were detected in 14%, 8%, and 18% of participants, respectively. CONCLUSIONS: HSV529 vaccine was safe and elicited neutralizing antibody and modest CD4+ T-cell responses in HSV-seronegative vaccinees. CLINICAL TRIALS REGISTRATION: NCT01915212.

Pharmacodynamics of anti-HIV gene therapy using viral vectors and targeted endonucleases.

OBJECTIVES: A promising curative approach for HIV is to use designer endonucleases that bind and cleave specific target sequences within latent genomes, resulting in mutations that render the virus replication incompetent. We developed a mathematical model to describe the expression and activity of endonucleases delivered to HIV-infected cells using engineered viral vectors in order to guide dose selection and predict therapeutic outcomes. METHODS: We developed a mechanistic model that predicts the number of transgene copies expressed at a given dose in individual target cells from fluorescence of a reporter gene. We fitted the model to flow cytometry datasets to determine the optimal vector serotype, promoter and dose required to achieve maximum expression. RESULTS: We showed that our model provides a more accurate measure of transduction efficiency compared with gating-based methods, which underestimate the percentage of cells expressing reporter genes. We identified that gene expression follows a sigmoid dose-response relationship and that the level of gene expression saturation depends on vector serotype and promoter. We also demonstrated that significant bottlenecks exist at the level of viral uptake and gene expression: only ∼1 in 220 added vectors enter a cell and, of these, depending on the dose and promoter used, between 1 in 15 and 1 in 1500 express transgene. CONCLUSIONS: Our model provides a quantitative method of dose selection and optimization that can be readily applied to a wide range of other gene therapy applications. Reducing bottlenecks in delivery will be key to reducing the number of doses required for a functional cure.

Detection of treatment-resistant infectious HIV after genome-directed antiviral endonuclease therapy.

Incurable chronic viral infections are a major cause of morbidity and mortality worldwide. One potential approach to cure persistent viral infections is via the use of targeted endonucleases. Nevertheless, a potential concern for endonuclease-based antiviral therapies is the emergence of treatment resistance. Here we detect for the first time an endonuclease-resistant infectious virus that is found with high frequency after antiviral endonuclease therapy. While testing the activity of HIV pol-specific zinc finger nucleases (ZFNs) alone or in combination with three prime repair exonuclease 2 (Trex2), we identified a treatment-resistant and infectious mutant virus that was derived from a ZFN-mediated disruption of reverse transcriptase (RT). Although gene disruption of HIV protease, RT and integrase could inhibit viral replication, a chance single amino acid insertion within the thumb domain of RT produced a virus that could actively replicate. The endonuclease-resistant virus could replicate in primary CD4(+) T cells, but remained susceptible to treatment with antiretroviral RT inhibitors. When secondary ZFN-derived mutations were introduced into the mutant virus's RT or integrase domains, replication could be abolished. Our observations suggest that caution should be exercised during endonuclease-based antiviral therapies; however, combination endonuclease therapies may prevent the emergence of resistance.