An overview of tecovirimat for smallpox treatment and expanded anti-orthopoxvirus applications.

INTRODUCTION: Tecovirimat (TPOXX®; ST-246) was approved for the treatment of symptomatic smallpox by the USFDA in July of 2018 and has been stockpiled by the US government for use in a smallpox outbreak. While there has not been a reported case of smallpox since 1978 it is still considered a serious bioterrorism threat. AREAS COVERED: A brief history of smallpox from its proposed origins as a human disease through its eradication in the late 20th century is presented. The current smallpox threat and the current public health response plans are described. The discovery, and development of tecovirimat through NDA submission and subsequent approval for treatment of smallpox are discussed. Google Scholar and PubMed were searched over all available dates for relevant publications. EXPERT OPINION: Approval of tecovirimat to treat smallpox represents an important milestone in biosecurity preparedness. Incorporating tecovirimat into the CDC smallpox response plan, development of pediatric liquid and intravenous formulations, and approval for post-exposure prophylaxis would provide additional health security benefit.Tecovirimat shows broad efficacy against orthopoxviruses in vitro and in vivo and could be developed for use against emerging orthopoxvirus diseases such as monkeypox, vaccination-associated adverse events, and side effects of vaccinia oncolytic virus therapy.

Oral Tecovirimat for the Treatment of Smallpox.

BACKGROUND: Smallpox was declared eradicated in 1980, but variola virus (VARV), which causes smallpox, still exists. There is no known effective treatment for smallpox; therefore, tecovirimat is being developed as an oral smallpox therapy. Because clinical trials in a context of natural disease are not possible, an alternative developmental path to evaluate efficacy and safety was needed. METHODS: We investigated the efficacy of tecovirimat in nonhuman primate (monkeypox) and rabbit (rabbitpox) models in accordance with the Food and Drug Administration (FDA) Animal Efficacy Rule, which was interpreted for smallpox therapeutics by an expert advisory committee. We also conducted a placebo-controlled pharmacokinetic and safety trial involving 449 adult volunteers. RESULTS: The minimum dose of tecovirimat required in order to achieve more than 90% survival in the monkeypox model was 10 mg per kilogram of body weight for 14 days, and a dose of 40 mg per kilogram for 14 days was similarly efficacious in the rabbitpox model. Although the effective dose per kilogram was higher in rabbits, exposure was lower, with a mean steady-state maximum, minimum, and average (mean) concentration (Cmax, Cmin, and Cavg, respectively) of 374, 25, and 138 ng per milliliter, respectively, in rabbits and 1444, 169, and 598 ng per milliliter in nonhuman primates, as well as an area under the concentration-time curve over 24 hours (AUC0-24hr) of 3318 ng×hours per milliliter in rabbits and 14,352 ng×hours per milliliter in nonhuman primates. These findings suggested that the nonhuman primate was the more conservative model for the estimation of the required drug exposure in humans. A dose of 600 mg twice daily for 14 days was selected for testing in humans and provided exposures in excess of those in nonhuman primates (mean steady-state Cmax, Cmin, and Cavg of 2209, 690, and 1270 ng per milliliter and AUC0-24hr of 30,632 ng×hours per milliliter). No pattern of troubling adverse events was observed. CONCLUSIONS: On the basis of its efficacy in two animal models and pharmacokinetic and safety data in humans, tecovirimat is being advanced as a therapy for smallpox in accordance with the FDA Animal Rule. (Funded by the National Institutes of Health and the Biomedical Advanced Research and Development Authority; ClinicalTrials.gov number, NCT02474589 .).

Novel benzoxazole inhibitor of dengue virus replication that targets the NS3 helicase.

Dengue virus (DENV) is the predominant mosquito-borne viral pathogen that infects humans with an estimated 50 to 100 million infections per year worldwide. Over the past 50 years, the incidence of dengue disease has increased dramatically and the virus is now endemic in more than 100 countries. Moreover, multiple serotypes of DENV are now found in the same geographic region, increasing the likelihood of more severe forms of disease. Despite extensive research, there are still no approved vaccines or therapeutics commercially available to treat DENV infection. Here we report the results of a high-throughput screen of a chemical compound library using a whole-virus assay that identified a novel small-molecule inhibitor of DENV, ST-610, that potently and selectively inhibits all four serotypes of DENV replication in vitro. Sequence analysis of drug-resistant virus isolates has identified a single point mutation, A263T, in the NS3 helicase domain that confers resistance to this compound. ST-610 inhibits DENV NS3 helicase RNA unwinding activity in a molecular-beacon-based helicase assay but does not inhibit nucleoside triphosphatase activity based on a malachite green ATPase assay. ST-610 is nonmutagenic, is well tolerated (nontoxic) in mice, and has shown efficacy in a sublethal murine model of DENV infection with the ability to significantly reduce viremia and viral load compared to vehicle controls.

SAR analysis of a series of acylthiourea derivatives possessing broad-spectrum antiviral activity.

A series of acylthiourea derivatives were designed, synthesized, and evaluated for broad-spectrum antiviral activity with selected viruses from Poxviridae (vaccinia virus) and two different genera of the family Bunyaviridae (Rift Valley fever and La Crosse viruses). A compound selected from a library screen, compound 1, displayed submicromolar antiviral activity against both vaccinia virus (EC(50)=0.25 µM) and La Crosse virus (EC(50)=0.27 µM) in cytopathic effect (CPE) assays. SAR analysis was performed to further improve antiviral potency and to optimize drug-like properties of the initial hits. During our analysis, we identified 26, which was found to be nearly fourfold more potent than 1 against both vaccinia and La Crosse viruses. Selected compounds were further tested to more fully characterize the spectrum of antiviral activity. Many of these possessed single digit micromolar and sub-micromolar antiviral activity against a diverse array of targets, including influenza virus (Orthomyxoviridae), Tacaribe virus (Arenaviridae), and dengue virus (Flaviviridae).

Cationic phosphorodiamidate morpholino oligomers efficiently prevent growth of Escherichia coli in vitro and in vivo.

OBJECTIVES: Phosphorodiamidate morpholino oligomers (PMOs) are uncharged DNA analogues that can inhibit bacterial growth by a gene-specific, antisense mechanism. Attaching cationic peptides to PMOs enables efficient penetration through the Gram-negative outer membrane. We hypothesized that cationic groups attached directly to the PMO would obviate the need to attach peptides. METHODS: PMOs with identical 11-base sequence (AcpP) targeted to acpP (an essential gene) of Escherichia coli were synthesized with various numbers of either piperazine (Pip) or N-(6-guanidinohexanoyl)piperazine (Gux) coupled to the phosphorodiamidate linker. Peptide-PMO conjugates were made using the membrane-penetrating peptide (RXR)(4)XB (X is 6-aminohexanoic acid; B is beta-alanine). RESULTS: MICs (microM/mg/L) were measured using E. coli: 3 + Pip-AcpP, 160/653; 6 + Pip-AcpP, 160/673; 2 + Gux-AcpP, 20/88; 5 + Gux-AcpP, 10/49; 8 + Gux-AcpP, 10/56; 3 + Pip-AcpP-(RXR)(4)XB, 0.3/2; and 5 + Gux-AcpP-(RXR)(4)XB, 0.6/4. In cell-free protein synthesis reactions, all PMOs inhibited gene expression approximately the same. These results suggested that Pip-PMOs inefficiently penetrated the outer membrane. Indeed, the MICs of 3 + Pip-AcpP and 6 + Pip-AcpP were reduced to 0.6 and 2.5 microM (1.2 and 10.5 mg/L), respectively, using as indicator a strain with a 'leaky' outer membrane. In vivo, mice were infected intraperitoneally with E. coli. Intraperitoneal treatment with 50 mg/kg 3 + Pip-AcpP, 15 mg/kg 5 + Gux-AcpP or 0.5 mg/kg 3 + Pip-AcpP-(RXR)(4)XB, or subcutaneous treatment with 15 mg/kg 5 + Gux-AcpP or (RXR)(4)XB-AcpP reduced bacteria in blood and increased survival. CONCLUSIONS: Cationic PMOs inhibited bacterial growth in vitro and in vivo, and Gux-PMOs were more effective than Pip-PMOs. However, neither was as effective as the equivalent PMO-peptide conjugates. Subcutaneous treatment showed that 5 + Gux-AcpP or (RXR)(4)XB-AcpP entered the circulatory system, reduced infection and increased survival.

Chemical modifications of antisense morpholino oligomers enhance their efficacy against Ebola virus infection.

Phosphorodiamidate morpholino oligomers (PMOs) are uncharged nucleic acid-like molecules designed to inactivate the expression of specific genes via the antisense-based steric hindrance of mRNA translation. PMOs have been successful at knocking out viral gene expression and replication in the case of acute viral infections in animal models and have been well tolerated in human clinical trials. We propose that antisense PMOs represent a promising class of therapeutic agents that may be useful for combating filoviral infections. We have previously shown that mice treated with a PMO whose sequence is complementary to a region spanning the start codon of VP24 mRNA were protected against lethal Ebola virus challenge. In the present study, we report on the abilities of two additional VP24-specific PMOs to reduce the cell-free translation of a VP24 reporter, to inhibit the in vitro replication of Ebola virus, and to protect mice against lethal challenge when the PMOs are delivered prior to infection. Additionally, structure-activity relationship evaluations were conducted to assess the enhancement of antiviral efficacy associated with PMO chemical modifications that included conjugation with peptides of various lengths and compositions, positioning of conjugated peptides to either the 5' or the 3' terminus, and the conferring of charge modifications by the addition of piperazine moieties. Conjugation with arginine-rich peptides greatly enhanced the antiviral efficacy of VP24-specific PMOs in infected cells and mice during lethal Ebola virus challenge.

Cell-penetrating peptides as transporters for morpholino oligomers: effects of amino acid composition on intracellular delivery and cytotoxicity.

Arginine-rich cell-penetrating peptides (CPPs) are promising transporters for intracellular delivery of antisense morpholino oligomers (PMO). Here, we determined the effect of L-arginine, D-arginine and non-alpha amino acids on cellular uptake, splice-correction activity, cellular toxicity and serum binding for 24 CPP-PMOs. Insertion of 6-aminohexanoic acid (X) or beta-alanine (B) residues into oligoarginine R8 decreased the cellular uptake but increased the splice-correction activity of the resulting compound, with a greater increase for the sequences containing more X residues. Cellular toxicity was not observed for any of the conjugates up to 10 microM. Up to 60 microM, only the conjugates with > or = 5 Xs exhibited time- and concentration-dependent toxicity. Substitution of L-arginine with D-arginine did not increase uptake or splice-correction activity. High concentration of serum significantly decreased the uptake and splice-correction activity of oligoarginine conjugates, but had much less effect on the conjugates containing X or B. In summary, incorporation of X/B into oligoarginine enhanced the antisense activity and serum-binding profile of CPP-PMO. Toxicity of X/B-containing conjugates was affected by the number of Xs, treatment time and concentration. More active, stable and less toxic CPPs can be designed by optimizing the position and number of R, D-R, X and B residues.