Novel Dengue Virus NS2B/NS3 Protease Inhibitors

Dengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PR in vitro and in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC₅₀s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro assays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC₅₀s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations.     

Targeting Organic Anion Transporter 3 with Probenecid as a Novel Anti-Influenza A Virus Strategy

Influenza A virus infection is a major global health concern causing significant mortality, morbidity, and economic loss. Antiviral chemotherapeutics that target influenza A virus are available; however, rapid emergence of drug-resistant strains has been reported. Consequently, there is a burgeoning need to identify novel anti-influenza A drugs, particularly those that target host gene products required for virus replication, to reduce the likelihood of drug resistance. In this study, a small interfering RNA (siRNA) screen was performed to identify host druggable gene targets for anti-influenza A virus therapy. The host organic anion transporter-3 gene (OAT3), a member of the SLC22 family of transporters, was validated as being required to support influenza A virus replication. Probenecid, a prototypical uricosuric agent and chemical inhibitor of organic anion transporters known to target OAT3, was shown to be effective in limiting influenza A virus infection in vitro (50% inhibitory concentration [IC₅₀] of 5.0 × 10⁻⁵ to 5.0 × 10⁻⁴ μM; P < 0.005) and in vivo (P < 0.05). Probenecid is widely used for treatment of gout and related hyperuricemic disorders, has been extensively studied for pharmacokinetics and safety, and represents an excellent candidate for drug repositioning as a novel anti-influenza A chemotherapeutic.     

In Silico Approach for Designing Potent Inhibitors Against Polymerase PB2 (Influenza A Virus: H1N1)

A 759 amino acid long sequence of polymerase PB2 [Influenza A virus {A/Mexico/47N/2009(H1N1)}] was taken to build the model of the protein which was validated by different tools of SAVS (structural analysis and verification server). The modeled protein was interacted with four different drugs available in the market viz. oseltaminivir, amantadine, zanasmivir, rimantadine (RS) and some naturally occurring compounds i.e. curcuminoids (curcumin, demethoxy curcumin, bis-demethoxy curcumin and cyclocurcumin) along with two synthetic bioconjugates of curcumin viz. dipiperoyl and disalicyloyl esters. The analysis parameters including docking score, reranking score and number of H-bonds indicate cyclocurcumin as the most favoured.     

A Novel Target and Approach for Identifying Antivirals against Molluscum Contagiosum Virus

The dermatological disease molluscum contagiosum (MC) presents as lesions restricted solely to the skin. The poxvirus molluscum contagiosum virus (MCV) is responsible for this skin disease that is easily transmitted through casual contact among all populations, with greater frequency in children and immunosuppressed individuals. In addition, sexual transmission of MCV in adolescents and adults is a health concern. Although the skin lesions ultimately resolve in immunocompetent individuals, they can persist for extended periods, be painful, and result in scarring. Treatment is problematic, and there is no drug that specifically targets MCV. The inability of MCV to propagate in cell culture has impeded drug development. To overcome these barriers, we integrated three new developments. First, we identified a new MCV drug target (mD4) that is essential for processive DNA synthesis in vitro. Second, we discovered a small chemical compound that binds to mD4 and prevents DNA synthesis in vitro. Third, and most significant, we engineered a hybrid vaccinia virus (mD4-VV) in which the natural vaccinia D4 (vD4) gene is replaced by the mD4 target gene. This hybrid virus is dependent on mD4 for viral growth in culture and is inhibited by the small compound. This target system provides, for the first time, a platform and approach for the discovery and evaluation of new therapeutics that can be used to treat MC.     

Treatment With Cyclooxygenase-2 Inhibitors Enables Repeated Administration of Vaccinia Virus for Control of Ovarian Cancer

Metastatic ovarian cancer is the leading cause of death among women with gynecologic malignancies in the United States. The lack of effective treatment for patients with advanced ovarian cancer warrants development of innovative therapies. Cancer therapy using oncolytic viruses represents a promising new approach for controlling tumors. Vaccinia virus has been shown to preferentially infect tumor cells but not normal tissue. However, oncolytic therapy using recombinant viruses faces the limitation of viral clearance due to generation of neutralizing antibodies. In the current study, we found that cyclooxygenase-2 (Cox-2) inhibitors circumvented this limitation, enabling repeated administration of vaccinia virus without losing infectivity. We quantified the antivaccinia antibody response using enzyme-linked immunosorbent assay (ELISA) and neutralization assays to show that treatment of Cox-2 inhibitors inhibited the generation of neutralizing antibodies. Furthermore, we showed that combination treatment of Cox-2 inhibitors with vaccinia virus was more effective that either treatment alone in treating MOSEC/luc tumor-bearing mice. Thus, the combination of Cox-2 inhibitors and vaccinia virus represents a potential innovative approach to controlling ovarian tumors.     

Novel S-Adenosylmethionine Decarboxylase Inhibitors for the Treatment of Human African Trypanosomiasis

Trypanosomiasis remains a significant disease across the sub-Saharan African continent, with 50,000 to 70,000 individuals infected. The utility of current therapies is limited by issues of toxicity and the need to administer compounds intravenously. We have begun a program to pursue lead optimization around MDL 73811, an irreversible inhibitor of S-adenosylmethionine decarboxylase (AdoMetDC). This compound is potent but in previous studies cleared rapidly from the blood of rats (T. L. Byers, T. L. Bush, P. P. McCann, and A. J. Bitonti, Biochem. J. 274:527-533). One of the analogs synthesized (Genz-644131) was shown to be highly active against Trypanosoma brucei rhodesiense in vitro (50% inhibitory concentration, 400 pg/ml). Enzyme kinetic studies showed Genz-644131 to be approximately fivefold more potent than MDL 73811 against the T. brucei brucei AdoMetDC-prozyme complex. This compound was stable in vitro in rat and human liver microsomal and hepatocyte assays, was stable in rat whole-blood assays, did not significantly inhibit human cytochrome P450 enzymes, had no measurable efflux in CaCo-2 cells, and was only 41% bound by serum proteins. Pharmacokinetic studies of mice following intraperitoneal dosing showed that the half-life of Genz-644131 was threefold greater than that of MDL 73811 (7.4 h versus 2.5 h). Furthermore, brain penetration of Genz-644131 was 4.3-fold higher than that of MDL 73811. Finally, in vivo efficacy studies of T. b. brucei strain STIB 795-infected mice showed that Genz-644131 significantly extended survival (from 6.75 days for controls to >30 days for treated animals) and cured animals infected with T. b. brucei strain LAB 110 EATRO. Taken together, the data strengthen validation of AdoMetDC as an important parasite target, and these studies have shown that analogs of MDL 73811 can be synthesized with improved potency and brain penetration.Sleeping sickness, or human African trypanosomiasis (HAT), afflicts 50,000 to 70,000 people across sub-Saharan Africa, with 17,000 new cases reported in the year 2004 (9) and 10,769 reported in 2007 (31). Untreated, the disease is inevitably fatal. Current treatments include drugs first developed over 50 years ago, and while not without efficacy, some have high toxicity and generally need to be administered by intravenous (i.v.) infusion—hardly a practical solution in locations where this disease is prevalent (17). Despite the obvious need for new, easily administered therapies, the rate of development of new drugs for HAT by the pharmaceutical industry has been negligible. Recognizing this, the Drugs for Neglected Diseases Initiative (DNDi) was formed in 2003 to facilitate the formation of partnerships among industry, academia, and public-sector organizations to develop affordable solutions for this urgent unmet medical need (www.dndi.org). Studies presented here were conducted under one such partnership.Polyamines are small-molecule cationic structures that are critical to the survival of eukaryotic cells, including trypanosomes (2, 4, 19). Difluoromethyl ornithine (DFMO) is an inhibitor of ornithine decarboxylase, a key enzyme in the polyamine biosynthetic pathway. DFMO is an effective and relatively well tolerated agent for the treatment of the central nervous system (CNS) second stage of HAT caused by Trypanosoma brucei gambiense. However, DFMO must be given as an i.v. infusion of up to 30 g per day four times per day for 14 days. The amount required and frequency of dosing demonstrate the limitations of this drug in terms of potency, cost, and logistics of administration in the field (10, 16). Nevertheless, the extraordinary efficacy of DFMO serves to validate the polyamine pathway as an attractive target for new drug discovery for trypanosomiasis. A potent, safe, orally bioavailable inhibitor of polyamine biosynthesis would represent a major breakthrough for the treatment of this disease.A second key enzyme in the polyamine biosynthetic pathway is S-adenosylmethionine decarboxylase (AdoMetDC). AdoMetDC is allosterically activated by a novel mechanism unique to trypanosomatid parasites; the functional form of the enzyme is a heterodimer between the active subunit and a paralog (termed prozyme) that arose through gene duplication (32). Gene knockout and small interfering RNA experiments have indicated that suppression of this enzyme by knockdown of either AdoMetDC or its regulatory subunit prozyme is lethal to the parasite (33). Inhibitors of this enzyme have been shown to be highly efficacious in killing trypanosomes in vitro (3, 14) and in curing T. brucei-infected mice (6, 8). However, these agents lack both the potency and, especially, the pharmacokinetic (PK) and tissue (CNS) distribution characteristics (11) that are essential to meet the improved target product profile for a new antitrypanosomal drug.MDL 73811 is an irreversible inhibitor of AdoMetDC and is believed to form a Schiff base with a pyruvate group within the active site of the enzyme (13). The compound displays a high level of selectivity (>100-fold) for killing parasites compared with its toxicity for mammalian cells (24). Reasons for this selectivity are not clear, but it has been postulated that the compound is more readily taken up by the parasites (12). Alternatively, selectivity may result from differences in enzyme turnover (33). The mechanism of killing is believed to be associated with the buildup of S-adenosylmethionine (11). Recent studies utilizing RNA interference silencing, however, suggest that a major method by which AdoMetDC inhibition kills trypanosomes is by depleting reserves of trypanothione (33). Finally, MDL 73811 induces increased expression of the prozyme in T. brucei bloodstream form parasites, providing strong evidence that the primary target of MDL 73811 responsible for parasite death is AdoMetDC inhibition (33). MDL 73811 has been demonstrated to reduce parasitemia within 5 h and to effect cures of acute infections in T. brucei brucei- and T. brucei rhodesiense-infected mice when administered at a dose of 20 mg/kg of body weight twice a day (BID) for 4 days (11, 24). This compound was originally developed in the 1980s by Merrell-Dow as an anticancer therapy.Despite its trypanocidal activity, MDL 73811 is not itself an attractive drug candidate for a number of reasons. This compound is not effective as monotherapy against the CNS stage of infection, although it is curative when given in combination with DFMO (6). It has been hypothesized that DFMO may in some way temporarily alter the blood-brain barrier (BBB) to effect this; however, data to support this contention are currently lacking. Recent studies (28) showed that DFMO by itself penetrated the BBB poorly and that addition of 250 μM DFMO did not improve the uptake of other solutes, although a statistically significant increase in BBB penetration was seen at day 28 and later after the trypanosome infection. Finally, the drug has poor oral bioavailability and was thought to have limited metabolic stability (P. Casara, personal communication). We therefore initiated a program to synthesize analogs of MDL 73811 and determine whether they could overcome these issues. Studies reported here describe initial progress.     

Preclinical Activity of VX-787, a First-in-Class,Orally Bioavailable Inhibitor of the Influenza Virus Polymerase PB2 Subunit

VX-787 is a novel inhibitor of influenza virus replication that blocks the PB2 cap-snatching activity of the influenza viral polymerase complex. Viral genetics and X-ray crystallography studies provide support for the idea that VX-787 occupies the 7-methyl GTP (m⁷GTP) cap-binding site of PB2. VX-787 binds the cap-binding domain of the PB2 subunit with a K_D (dissociation constant) of 24 nM as determined by isothermal titration calorimetry (ITC). The cell-based EC₅₀ (the concentration of compound that ensures 50% cell viability of an uninfected control) for VX-787 is 1.6 nM in a cytopathic effect (CPE) assay, with a similar EC₅₀ in a viral RNA replication assay. VX-787 is active against a diverse panel of influenza A virus strains, including H1N1pdm09 and H5N1 strains, as well as strains with reduced susceptibility to neuraminidase inhibitors (NAIs). VX-787 was highly efficacious in both prophylaxis and treatment models of mouse influenza and was superior to the neuraminidase inhibitor, oseltamivir, including in delayed-start-to-treat experiments, with 100% survival at up to 96 h postinfection and partial survival in groups where the initiation of therapy was delayed up to 120 h postinfection. At different doses, VX-787 showed a 1-log to >5-log reduction in viral load (relative to vehicle controls) in mouse lungs. Overall, these favorable findings validate the PB2 subunit of the viral polymerase as a drug target for influenza therapy and support the continued development of VX-787 as a novel antiviral agent for the treatment of influenza infection.     

Important Dietary Considerations for Sodium-Glucose Cotransporter 2 Inhibitors

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a pharmacologic option for type 2 diabetes, and several benefits beyond glycemic lowering, including cardiovascular and renal, are associated with their use. However, some rare but serious potential adverse effects have been reported, including diabetic ketoacidosis (DKA) and euglycemic DKA. Certain dietary factors may increase the risk of SGLT2 inhibitor–associated ketoacidosis, such as low-carbohydrate and ketogenic diets, prolonged fasting, dehydration, and excess alcohol consumption. Clinicians should remain cognizant of precipitating factors; discuss modifiable risk factors with patients; and implement preventive strategies, such as withholding a SGLT2 inhibitor in situations associated with risk.     

Novel Cell-Based Hepatitis C Virus Infection Assay for Quantitative High-Throughput Screening of Anti-Hepatitis C Virus Compounds

Therapy for hepatitis C virus (HCV) infection has advanced with the recent approval of direct-acting antivirals in combination with peginterferon and ribavirin. New antivirals with novel targets are still needed to further improve the treatment of hepatitis C. Previously reported screening methods for HCV inhibitors either are limited to a virus-specific function or apply a screening method at a single dose, which usually leads to high false-positive or -negative rates. We developed a quantitative high-throughput screening (qHTS) assay platform with a cell-based HCV infection system. This highly sensitive assay can be miniaturized to a 1,536-well format for screening of large chemical libraries. All candidates are screened over a 7-concentration dose range to give EC₅₀s (compound concentrations at 50% efficacy) and dose-response curves. Using this assay format, we screened a library of pharmacologically active compounds (LOPAC). Based on the profile of dose-dependent curves of HCV inhibition and cytotoxicity, 22 compounds with adequate curves and EC₅₀s of <10 μM were selected for validation. In two additional independent assays, 17 of them demonstrated specific inhibition of HCV infection. Ten potential candidates with efficacies of >70% and CC₅₀s (compound concentrations at 50% cytotoxicity) of <30 μM from these validated hits were characterized for their target stages in the HCV replication cycle. In this screen, we identified both known and novel hits with diverse structural and functional features targeting various stages of the HCV replication cycle. The pilot screen demonstrates that this assay system is highly robust and effective in identifying novel HCV inhibitors and that it can be readily applied to large-scale screening of small-molecule libraries.