Antiviral Rotenoids and Isoflavones Isolated from Millettia oblata ssp. teitensis.

Three new (1-3) and six known rotenoids (5-10), along with three known isoflavones (11-13), were isolated from the leaves of Millettia oblata ssp. teitensis. A new glycosylated isoflavone (4), four known isoflavones (14-18), and one known chalcone (19) were isolated from the root wood extract of the same plant. The structures were elucidated by NMR and mass spectrometric analyses. The absolute configuration of the chiral compounds was established by a comparison of experimental ECD and VCD data with those calculated for the possible stereoisomers. This is the first report on the use of VCD to assign the absolute configuration of rotenoids. The crude leaves and root wood extracts displayed anti-RSV (human respiratory syncytial virus) activity with IC50 values of 0.7 and 3.4 µg/mL, respectively. Compounds 6, 8, 10, 11, and 14 showed anti-RSV activity with IC50 values of 0.4-10 µM, while compound 3 exhibited anti-HRV-2 (human rhinovirus 2) activity with an IC50 of 4.2 µM. Most of the compounds showed low cytotoxicity for laryngeal carcinoma (HEp-2) cells; however compounds 3, 11, and 14 exhibited low cytotoxicity also in primary lung fibroblasts. This is the first report on rotenoids showing antiviral activity against RSV and HRV viruses.

Identification of epidermal growth factor receptor-tyrosine kinase inhibitor targeting the VP1 pocket of human rhinovirus.

Screening a library of 1,200 preselected kinase inhibitors for anti-human rhinovirus 2 (HRV-2) activity in HeLa cells identified a class of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) as effective virus blockers. These were based on the 4-anilinoquinazoline-7-oxypiperidine scaffold, with the most potent representative AZ5385 inhibiting the virus with EC50 of 0.35 µM. Several structurally related analogs confirmed activity in the low µM range, while interestingly, other TKIs targeting EGFR lacked anti-HRV-2 activity. To further probe this lack of association between antiviral activity and EGFR inhibition, we stained infected cells with antibodies specific for activated EGFR (Y1068) and did not observe a dependency on EGFR-TK activity. Instead, consecutive passages of HRV-2 in HeLa cells in the presence of a compound and subsequent nucleotide sequence analysis of resistant viral variants identified the S181T and T210A alterations in the major capsid VP1 protein, with both residues located in the vicinity of a known hydrophobic pocket on the viral capsid. Further characterization of the antiviral effects of AZ5385 showed a modest virus-inactivating (virucidal) activity, while anti-HRV-2 activity was still evident when the inhibitor was added as late as 10 h post infection. The RNA copy/infectivity ratio of HRV-2 propagated in AZ5385 presence was substantially higher than that of control HRV indicating that the compound preferentially targeted HRV progeny virions during their maturation in infected cells. Besides HRV, the compound showed anti-respiratory syncytial virus activity, which warrants its further studies as a candidate compound against viral respiratory infections.

Crotofolane Diterpenoids and Other Constituents Isolated from Croton kilwae.

Six new crotofolane diterpenoids (1-6) and 13 known compounds (7-19) were isolated from the MeOH-CH2Cl2 (1:1, v/v) extracts of the leaves and stem bark of Croton kilwae. The structures of the new compounds were elucidated by extensive analysis of spectroscopic and mass spectrometric data. The structure of crotokilwaepoxide A (1) was confirmed by single-crystal X-ray diffraction, allowing for the determination of its absolute configuration. The crude extracts and the isolated compounds were investigated for antiviral activity against respiratory syncytial virus (RSV) and human rhinovirus type-2 (HRV-2) in HEp-2 and HeLa cells, respectively, for antibacterial activity against the Gram-positive Bacillus subtilis and the Gram-negative Escherichia coli, and for antimalarial activity against the Plasmodium falciparum Dd2 strain. ent-3ß,19-Dihydroxykaur-16-ene (7) and ayanin (16) displayed anti-RSV activities with IC50 values of 10.2 and 6.1 µM, respectively, while exhibiting only modest cytotoxic effects on HEp-2 cells that resulted in selectivity indices of 4.9 and 16.4. Compounds 2 and 5 exhibited modest anti-HRV-2 activity (IC50 of 44.6 µM for both compounds), while compound 16 inhibited HRV-2 with an IC50 value of 1.8 µM. Compounds 1-3 showed promising antiplasmodial activities (80-100% inhibition) at a 50 µM concentration.

Modified ent-Abietane Diterpenoids from the Leaves of Suregada zanzibariensis.

The leaf extract of Suregada zanzibariensis gave two new modified ent-abietane diterpenoids, zanzibariolides A (1) and B (2), and two known triterpenoids, simiarenol (3) and ß-amyrin (4). The structures of the isolated compounds were elucidated based on NMR and MS data analysis. Single-crystal X-ray diffraction was used to establish the absolute configurations of compounds 1 and 2. The crude leaf extract inhibited the infectivity of herpes simplex virus 2 (HSV-2, IC50 11.5 µg/mL) and showed toxicity on African green monkey kidney (GMK AH1) cells at CC50 52 µg/mL. The isolated compounds 1-3 showed no anti-HSV-2 activity and exhibited insignificant toxicity against GMK AH1 cells at ≥100 µM.

Synthetic Heparan Sulfate Mimetic Pixatimod (PG545) Potently Inhibits SARS-CoV-2 by Disrupting the Spike-ACE2 Interaction.

Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a coreceptor with the ACE2 protein for the S1 spike protein on SARS-CoV-2 virus, providing a tractable new therapeutic target. Clinically used heparins demonstrate an inhibitory activity but have an anticoagulant activity and are supply-limited, necessitating alternative solutions. Here, we show that synthetic HS mimetic pixatimod (PG545), a cancer drug candidate, binds and destabilizes the SARS-CoV-2 spike protein receptor binding domain and directly inhibits its binding to ACE2, consistent with molecular modeling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of SARS-CoV-2 virus show that pixatimod potently inhibits the infection of monkey Vero E6 cells and physiologically relevant human bronchial epithelial cells at safe therapeutic concentrations. Pixatimod also retained broad potency against variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, in a K18-hACE2 mouse model, pixatimod significantly reduced SARS-CoV-2 viral titers in the upper respiratory tract and virus-induced weight loss. This demonstration of potent anti-SARS-CoV-2 activity tolerant to emerging mutations establishes proof-of-concept for targeting the HS-Spike protein-ACE2 axis with synthetic HS mimetics and provides a strong rationale for clinical investigation of pixatimod as a potential multimodal therapeutic for COVID-19.

Anti-respiratory syncytial virus and anti-herpes simplex virus activity of six Tanzanian medicinal plants with extended studies of Erythrina abyssinica stem bark.

ETHNOPHARMACOLOGICAL RELEVANCE: Except for few highly pathogenic viruses, no antiviral drug has been approved for treatment of viral infections in humans. Plant extracts, selected based on their ethno-medical use, represent an important source of compounds for the development of novel candidate antiviral drugs. This especially concerns plants with ethnomedical records on their use in treatment of viral infections. AIM OF THE STUDY: To identify and document medicinal plants used by traditional health practitioners (THPs) for treatment of respiratory infections and muco-cutaneous lesions in order to study their antiviral activity including identification of active components and elucidation of mode of antiviral activity. MATERIALS AND METHODS: The ethno-medical survey was performed in the Kagera region of Tanzania. The THPs were asked for plants used for treatment of signs and symptoms of respiratory infections and watery muco-cutaneous blisters in oral and genital regions. The plants identified were successively extracted with n-hexane, ethyl acetate and water, and the extracts assayed for anti-respiratory syncytial virus (RSV), anti-herpes simplex virus 2 (HSV-2), and anti-human parainfluenza virus 2 (HPIV-2) activity in cultured cells. Antiviral components were separated by ethanol precipitation and CL-6B chromatography, and the mode of antiviral activity elucidated by the time-of-addition assay and selection for the virus variants resistant to antiviral plant extract. RESULTS: THPs identified fifteen plants used for treatment of respiratory infections and muco-cutaneous blisters. The water extract, but not n-hexane or ethyl acetate extracts, of six of these plants including Erythrina abyssinica stem bark, inhibited infectivity of two glycosaminoglycan-binding viruses i.e., RSV and HSV-2 but not the sialic acid binding HPIV-2. An activity-guided separation revealed that antiviral component(s) of water extract of E. abyssinica could be precipitated with ethanol. This sample potently and selectively inhibited RSV and HSV-2 infectivity in cultured cells with IC50 values of 2.1 µg/ml (selectivity index >476) and 0.14 µg/ml (selectivity index >7143) respectively. The sample exhibited inhibitory effect on the virus attachment to and entry into the cells by directly targeting the viral particles. Indeed, 10 consecutive virus passages in HEp-2 cells in the presence of this extract selected for a resistant RSV variant lacking the attachment, viral membrane-associated, G protein due to a stop codon at amino acid residue 33 (Leu33stop). Fractionation of the E. abyssinica extract on a CL-6B column revealed that anti-RSV and HSV-2 activity correlated with carbohydrate content. The most pronounced antiviral activity was associated with a carbohydrate containing ingredient of molecular mass of <5 kDa, which may polymerize to antiviral composites of up to 410 kDa. CONCLUSIONS: Altogether, the water extract of six medicinal plants showed anti-RSV and anti-HSV-2 activities. Extended studies of the stem bark of E. abyssinica identified antiviral components that potently and selectively inhibited infectivity of free RSV and HSV-2 particles, a feature of importance in topical treatment of these infections. This observation confirms ethno-medical information concerning the use of E. abyssinica extract for treatment of respiratory infections and herpetic lesions.

Antiviral iridoid glycosides from Clerodendrum myricoides.

The methanol root extract of Clerodendrum myricoides (Hochst.) Vatke afforded two new (1, 2) and two known (3, 4) iridoid glycosides. The structures of the isolated compounds were established based on NMR, IR, UV and MS data analyses. The crude extract and the isolated constituents were assayed for antiviral activity against the human respiratory syncytial virus (RSV) in human laryngeal epidermoid carcinoma (HEp-2) cells. The crude extract inhibited RSV infectivity at EC50 = 0.21 µg/ml, while it showed cytotoxicity against HEp-2 cells with CC50 = 9 µg/ml. Compound 2 showed 43.2% virus inhibition at 100 µM, while compounds 1 as well as 3 and 4 had only weak antiviral and cytotoxic activities.

High Frequency of Either Altered Pre-Core StartCodon or Weakened Kozak Sequence in the CorePromoter Region in Hepatitis B Virus A1 Strainsfrom Rwanda.

Hepatitis B virus (HBV) is endemic in Rwanda and is a major etiologic agent for chronic liver disease in the country. In a previous analysis of HBV strains from Rwanda, the S genes of most strains segregated into one single clade of subgenotype, A1. More than half (55%) of the anti-HBe positive individuals were viremic. In this study, 23 complete HBV genomes and the core promoter region (CP) from 18 additional strains were sequenced. Phylogenetic analysis of complete genomes confirmed that most Rwandan strain formed a single unique clade, within subgenotype A1. Strains from 17 of 22 (77%) anti-HBe positive HBV carriers had either mutated the precore start codon (9 strains with either CUG, ACG, UUG, or AAG) or mutations in the Kozak sequence preceding the pre-core start codon (8 strains). These mutually exclusive mutations were also identified in subgenotypes A1 (70/266; 26%), A2 (12/255; 5%), and A3 (26/49; 53%) sequences from the GenBank. The results showed that previous, rarely described HBV variants, expressing little or no HBeAg, are selected in anti-HBe positive subgenotype Al carriers from Rwanda and that mutations reducing HBeAg synthesis might be unique for a particular HBV clade, not just for a specific genotype or subgenotype.

Inosine Triphosphate Pyrophosphatase Dephosphorylates Ribavirin Triphosphate and Reduced Enzymatic Activity Potentiates Mutagenesis in Hepatitis C Virus.

A third of humans carry genetic variants of the ITP pyrophosphatase (ITPase) gene (ITPA) that lead to reduced enzyme activity. Reduced ITPase activity was earlier reported to protect against ribavirin-induced hemolytic anemia and to diminish relapse following ribavirin and interferon therapy for hepatitis C virus (HCV) genotype 2 or 3 infections. While several hypotheses have been put forward to explain the antiviral actions of ribavirin, details regarding the mechanisms of interaction between reduced ITPase activity and ribavirin remain unclear. The in vitro effect of reduced ITPase activity was assessed by means of transfection of hepatocytes (Huh7.5 cells) with a small interfering RNA (siRNA) directed against ITPA or a negative-control siRNA in the presence or absence of ribavirin in an HCV culture system. Low ribavirin concentrations strikingly depleted intracellular GTP levels in HCV-infected hepatocytes whereas higher ribavirin concentrations induced G-to-A and C-to-U single nucleotide substitutions in the HCV genome, with an ensuing reduction of HCV RNA expression and HCV core antigen production. Ribavirin triphosphate (RTP) was dephosphorylated in vitro by recombinant ITPase to a similar extent as ITP, a naturally occurring substrate of ITPase, and reducing ITPA expression in Huh 7.5 cells by siRNA increased intracellular levels of RTP in addition to increasing HCV mutagenesis and reducing progeny virus production. Our results extend the understanding of the biological impact of reduced ITPase activity, demonstrate that RTP is a substrate of ITPase, and may point to personalized ribavirin dosage according to ITPA genotype in addition to novel antiviral strategies.IMPORTANCE This study highlights the multiple modes of action of ribavirin, including depletion of intracellular GTP and increased hepatitis C virus mutagenesis. In cell culture, reduced ITP pyrophosphatase (ITPase) enzyme activity affected the intracellular concentrations of ribavirin triphosphate (RTP) and augmented the impact of ribavirin on the mutation rate and virus production. Additionally, our results imply that RTP, similar to ITP, a naturally occurring substrate of ITPase, is dephosphorylated in vitro by ITPase.

The Cholestanol-Conjugated Sulfated Oligosaccharide PG545 Disrupts the Lipid Envelope of Herpes Simplex Virus Particles.

Herpes simplex virus (HSV) and many other viruses, including HIV, initiate infection of host cells by binding to glycosaminoglycan (GAG) chains of cell surface proteoglycans. Although GAG mimetics, such as sulfated oligo- and polysaccharides, exhibit potent antiviral activities in cultured cells, the prophylactic application of these inhibitors as vaginal microbicides failed to protect women upon their exposure to HIV. A possible explanation for this failure is that sulfated oligo- and polysaccharides exhibit no typical virucidal activity, as their interaction with viral particles is largely electrostatic and reversible and thereby vulnerable to competition with GAG-binding proteins of the genital tract. Here we report that the cholestanol-conjugated sulfated oligosaccharide PG545, but not several other sulfated oligosaccharides lacking this modification, exhibited virucidal activity manifested as disruption of the lipid envelope of HSV-2 particles. The significance of the virus particle-disrupting activity of PG545 was also demonstrated in experimental animals, as this compound, in contrast to unmodified sulfated oligosaccharide, protected mice against genital infection with HSV-2. Thus, PG545 offers a novel prophylaxis option against infections caused by GAG-binding viruses.

Lipophile-conjugated sulfated oligosaccharides as novel microbicides against HIV-1.

With the aim of providing compounds suitable for further development as microbicides active against human immunodeficiency virus 1 (HIV-1) a library containing 37 lipophile-conjugated sulfated oligosaccharides was screened for antiviral and virucidal activity against this virus. Four highly active compounds had low drug inhibition concentrations (IC(50)) for HIV-1 and inactivated viral particles, suggestive of virucidal properties. Two of these compounds comprising a sulfated tetrasaccharide linked to a cholestanol group by a glycosidic bond, showed low toxicity and high selectivity indices. The two compounds were active both against CCR5 and dual-tropic CCR5/CXCR4 clinical HIV-1 isolates. Since herpes simplex virus type 2 (HSV-2) may be a cofactor for HIV-1 infection, the virucidal effect of the compounds was demonstrated against both viruses when mixed and incubated together on permissive cells. Incubation of compounds with serum, and to a lesser degree, cervical secretions, reduced the HIV-1 inactivating capacity, which suggests the need for molecular modification to reduce host protein binding. Considering the virucidal effect and low toxicity, these sulfated oligosaccharides with lipophilic tails may offer new possibilities of microbicide development.