Betulin Is a Potent Anti-Tumor Agent that Is Enhanced by Cholesterol

Betulinic Acid (BetA) and its derivatives have been extensively studied in the past for their anti-tumor effects, but relatively little is known about its precursor Betulin (BE). We found that BE induces apoptosis utilizing a similar mechanism as BetA and is prevented by cyclosporin A (CsA). BE induces cell death more rapidly as compared to BetA, but to achieve similar amounts of cell death a considerably higher concentration of BE is needed. Interestingly, we observed that cholesterol sensitized cells to BE-induced apoptosis, while there was no effect of cholesterol when combined with BetA. Despite the significantly enhanced cytotoxicity, the mode of cell death was not changed as CsA completely abrogated cell death. These results indicate that BE has potent anti-tumor activity especially in combination with cholesterol.     

In Vitro Antiviral Activity of V-073 against Polioviruses

V-073, an enterovirus capsid inhibitor, was evaluated for its spectrum of antipoliovirus activity. V-073 inhibited all 45 polioviruses tested in a virus-induced cytopathic effect protection assay, with 50% effective concentration (EC₅₀) values ranging from 0.003 to 0.126 μM. Ninety percent of the polioviruses tested were inhibited at EC₅₀s of ≤0.076 μM (MIC₉₀ = 32 ng/ml). V-073 is a promising antiviral candidate for the posteradication management of poliovirus incidents.     

Kibdelomycin Is a Potent and Selective Agent against Toxigenic Clostridium difficile

Clostridium difficile is the causative agent of C. difficile-associated diarrhea (CDAD), with increased risk in elderly populations. Kibdelomycin, a novel natural-product inhibitor of type II topoisomerase enzymes, was evaluated for activity against C. difficile and gastrointestinal anaerobic organisms. Toxigenic C. difficile isolates (n = 168) from U.S. hospitals and anaerobic Gram-positive and Gram-negative organisms (n = 598) from Chicago-area hospitals were tested. Kibdelomycin showed potent activity against toxigenic C. difficile (MIC₉₀ = 0.25 μg/ml) and most Gram-positive aerobic organisms but had little activity against Bacteroides species (MIC₅₀ > 32 μg/ml; n = 270). Potent anti-C. difficile activity was also observed in the hamster model of C. difficile colitis. Dosing at 1.6 mg/kg (twice-daily oral dose) resulted in protection from a lethal infection and a 2-log reduction in C. difficile cecal counts. A 6.25-mg/kg twice-daily oral dose completely eliminated detectable C. difficile counts in cecal contents. A single 6.25-mg/kg oral dose showed that cecal contents were exposed to the drug at >2 μM (eightfold higher than the MIC), with no significant plasma exposure. These findings support further exploration of kibdelomycin for development of an anti-C. difficile agent.     

Luliconazole Demonstrates Potent In Vitro Activity against Dermatophytes Recovered from Patients with Onychomycosis

The in vitro activities of luliconazole, amorolfine, ciclopirox, and terbinafine were determined against 320 dermatophyte isolates from large toenails of onychomycosis patients enrolled into an ongoing phase 2b/3 clinical study. The geometric mean MIC for luliconazole was 0.00022 μg/ml against all isolates, compared to 0.0194 to 0.3107 μg/ml for the three other agents. The in vitro potency of luliconazole was maintained regardless of the dermatophyte species.     

In Vitro Antiviral Activity and Preliminary Clinical Trials of a New Adamantane Compound

A compound, 1'-methyl spiro (adamantane-2,3'-pyrrolidine) maleate, chemically related to the antiviral drug amantadine, was tested for activity in vitro against a number of human respiratory viruses. By a variety of techniques, it was shown to be active against a wide range of human and animal influenza A viruses. The effect was, however, variable and ranged from high activity against two 1957 Asian strains to no observable activity against a 1971 strain. Like amantadine, the drug did not inhibit the growth of influenza B viruses. It was also inactive against a number of paramyxoviruses. Unlike amantadine, the drug did inhibit rhinoviruses, but to a lesser extent than myxoviruses. The coronavirus 229E was also sensitive to the action of the drug in vitro. Although an earlier trial in volunteers showed that, when given orally from 2 days before until 5 days after virus challenge, the drug was protective against infection with influenza A/Hong Kong/68 virus, a similar trial in volunteers challenged with rhinoviruses 2 and 9 revealed no useful activity against rhinoviruses in man.     

Syndecan-Fc Hybrid Molecule as a Potent In Vitro Microbicidal Anti-HIV-1 Agent

In the absence of a vaccine, there is an urgent need for the development of safe and effective topical microbicides to prevent the sexual transmission of human immunodeficiency virus type 1 (HIV-1). In this study, we proposed to develop a novel class of microbicides using syndecan as the antiviral agent. Specifically, we generated a soluble syndecan-Fc hybrid molecule by fusing the ectodomain of syndecan-1 to the Fc domain of a human IgG. We then tested the syndecan-Fc hybrid molecule for various in vitro microbicidal anti-HIV-1 properties. Remarkably, the syndecan-Fc hybrid molecule possesses multiple attractive microbicidal properties: (i) it blocks HIV-1 infection of primary targets including T cells, macrophages, and dendritic cells (DC); (ii) it exhibits a broad range of antiviral activity against primary HIV-1 isolates, multidrug resistant HIV-1 isolates, HIV-2, and simian immunodeficiency virus (SIV); (iii) it prevents transmigration of HIV-1 through human primary genital epithelial cells; (iv) it prevents HIV-1 transfer from dendritic cells to CD4⁺ T cells; (v) it is potent when added 2 h prior to addition of HIV-1 to target cells; (vi) it is potent at a low pH; (vii) it blocks HIV-1 infectivity when diluted in genital fluids; and (viii) it prevents herpes simplex virus infection. The heparan sulfate chains of the syndecan-Fc hybrid molecule are absolutely required for HIV-1 neutralization. Several lines of evidence suggest that the highly conserved Arg298 in the V3 region of gp120 serves as the locus for the syndecan-Fc hybrid molecule neutralization. In conclusion, this study suggests that the syndecan-Fc hybrid molecule represents the prototype of a new generation of microbicidal agents that may have promise for HIV-1 prevention.The dominant cell surface heparan sulfate proteoglycans (HSPG) (25, 29, 30, 33) are syndecans, which are transmembrane receptors highly expressed on adherent cells (macrophages and epithelial and endothelial cells) but poorly expressed on suspension cells (T cells) (2, 3, 4, 10, 35). Their ectodomain bears three linear heparan sulfate (HS) chains, which are composed of a repetition of a sulfated disaccharide motif (1). The sulfation pattern of HSs dictates the ligand specificity of syndecans (1). HSPG, including syndecans, serve as receptors for human deficiency virus type-1 (HIV-1) (16), herpes simplex virus (HSV) (7), human papillomavirus (HPV) (13, 37), and human T-lymphotropic virus type 1 (HTLV-1) (19, 20). Pretreatment of target cells such as macrophages with heparinase, an enzyme that removes HS moieties from syndecans, significantly reduces HIV-1 infectivity (35). Although syndecans do not alleviate the requirement for CD4 and chemokine receptors for viral entry (35), these in cis attachment receptors amplify HIV-1 infection by promoting viral adsorption to the surface of permissive cells. Syndecans also serve as in trans receptors for HIV-1 (2, 16). HIV-1 binds syndecans richly expressed on the endothelium and remains infectious for a week, whereas cell-free virus loses its infectivity after a single day (2). Moreover, HIV-1 attached onto the endothelium via syndecans represents an in trans source of infection for circulating T cells (2). Primary HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates produced from peripheral blood mononuclear cells (PBMCs) exploit syndecans (2). Furthermore, syndecans on microvascular endothelial cells play a significant role in cell-free HIV-1 transmigration through the blood-brain barrier (3). Thus, HIV-1 has maximized its utilization of syndecans in the body.A single conserved arginine (Arg298) in the V3 region of gp120 governs HIV-1 binding to syndecans (11). An amine group on the side chain of this residue is absolutely required for syndecan utilization by HIV-1 (11). HIV-1 binds syndecans via a 6-O sulfation (11) within the HS chains, demonstrating that this binding is not the result of random interactions between basic residues and negative charges but the result of specific contacts between gp120 and a well-defined sulfation in syndecans. Surprisingly, the Arg298 in gp120 that mediates HIV-1 binding to syndecans also mediates HIV-1 binding to CCR5 (42), suggesting that HIV-1 recognizes similar motifs on syndecans and CCR5 (11). Supporting this hypothesis, the 6-O sulfation recognized by HIV-1 on syndecans mimics the sulfated tyrosines recognized by HIV-1 in the N terminus of CCR5 (11). The finding that CCR5 and syndecans are exploited by HIV-1 via a single determinant echoes the mechanisms by which chemokines utilize these two disparate receptors and suggests that the gp120/chemokine mimicry may represent a common strategy in microbial pathogenesis.More recent work suggests that syndecans play a critical role in HIV-1 transmission (4). HIV-1 transmission includes transmigration of HIV-1 through the genital epithelium and subsequent capture and transfer of infectious particles from dendritic cells (DC) and/or Langerhans cells (LC) to T cells (31, 34, 38). Importantly, human cervical and vaginal mucosal epithelia richly express syndecans in vivo (4). HS chain removal by heparinase treatment prevents HIV-1 transcytosis through primary human cervical and vaginal mucosal epithelia (4). The Arg298 V3 HIV-1 mutant, which cannot interact with syndecans (11), fails to transcytose (4). Thus, syndecans facilitate HIV-1 epithelial transmigration in vitro. More recently, we identified syndecan-3 as an HIV-1 receptor on DC (12). Syndecan-3 stabilizes the captured virus, enhances DC infection in cis, and promotes transfer to T cells (12). Removal of the HS from the cell surface by heparinase or by silencing syndecan-3 by small interfering RNA (siRNA) partially inhibited HIV-1 transmission by immature DC (12), whereas neutralizing both syndecan-3 and DC-SIGN abrogated HIV-1 capture and subsequent transmission (12). Our findings that syndecans modulate both HIV-1 epithelial transmigration and HIV-1 transfer from DC to CD4⁺ T cells suggest that syndecans represent new targets for the development of topical microbicides.Nearly 40 million people currently live with HIV-1/AIDS; the majority of them reside in developing countries. It has been estimated that there are over 14,000 new HIV-1 infections per day in adults aged 15 to 50 years, with 50% of the newly infected being women aged 15 to 24 years (21, 22). In the absence of a vaccine, there is thus an urgent need for the development of safe and effective topical microbicides to prevent the sexual transmission of HIV-1 (14, 26). Topical microbicides are currently defined as vaginally or rectally applied products that prevent male-to-female or female-to-male transmission of HIV-1 infectious particles (39).In this study, we proposed to develop a novel class of microbicides using syndecan as the antiviral agent. Specifically, we generated a soluble syndecan-Fc hybrid molecule by fusing the ectodomain of syndecan-1 to the Fc domain of a human IgG. We then tested this syndecan-Fc hybrid molecule for its in vitro microbicidal properties. Remarkably, the syndecan-Fc hybrid molecule possesses multiple attractive microbicidal properties: (i) it blocks HIV-1 infection of primary targets including T cells, macrophages, and DC; (ii) it exhibits a broad range of antiviral activity against primary HIV-1 isolates, multidrug-resistant HIV-1 isolates, HIV-1, and SIV; (iii) it prevents transmigration of HIV-1 through human primary genital epithelial cells (PGEC); (iv) it prevents HIV-1 transfer from DC to CD4⁺ T cells; (v) it is potent when added 2 h prior to addition of HIV-1 to target cells; (vi) it is potent at a low pH; (vii) it blocks HIV-1 infectivity when diluted in genital fluids; and (viii) it inhibits HSV infection. Thus, this syndecan-Fc hybrid molecule represents the prototype of a new generation of microbicidal agents that may have promise for HIV-1 prevention.     

Identification of 23-(S)-2-Amino-3-Phenylpropanoyl-Silybin as an Antiviral Agent for Influenza A Virus Infection In Vitro and In Vivo

It has been reported that autophagy is involved in the replication of many viruses. In this study, we screened 89 medicinal plants, using an assay based on the inhibition of the formation of the Atg12-Atg5/Atg16 heterotrimer, an important regulator of autophagy, and selected Silybum marianum L. for further study. An antiviral assay indicated that silybin (S0), the major active compound of S. marianum L., can inhibit influenza A virus (IAV) infection. We later synthesized 5 silybin derivatives (S1 through S5) and found that 23-(S)-2-amino-3-phenylpropanoyl-silybin (S3) had the best activity. When we compared the polarities of the substituent groups, we found that the hydrophobicity of the substituent groups was positively correlated with their activities. We further studied the mechanisms of action of these compounds and determined that S0 and S3 also inhibited both the formation of the Atg12-Atg5/Atg16 heterotrimer and the elevated autophagy induced by IAV infection. In addition, we found that S0 and S3 could inhibit several components induced by IAV infection, including oxidative stress, the activation of extracellular signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase (MAPK) and IκB kinase (IKK) pathways, and the expression of autophagic genes, especially Atg7 and Atg3. All of these components have been reported to be related to the formation of the Atg12-Atg5/Atg16 heterotrimer, which might validate our screening strategy. Finally, we demonstrated that S3 can significantly reduce influenza virus replication and the associated mortality in infected mice. In conclusion, we identified 23-(S)-2-amino-3-phenylpropanoyl-silybin as a promising inhibitor of IAV infection.     

Ceftaroline plus Avibactam Demonstrates Bactericidal Activity against Pathogenic Anaerobic Bacteria in a One-Compartment In Vitro Pharmacokinetic/Pharmacodynamic Model

Anaerobic pathogens are often associated with polymicrobial infections, such as diabetic foot infections. Patients with these infections are often treated with broad-spectrum, multidrug therapies targeting resistant Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus, as well as Gram-negative bacteria and anaerobes. The broad-spectrum, non-beta-lactam, beta-lactamase inhibitor avibactam has been combined with ceftaroline and may provide a single-product alternative for complicated polymicrobial infections. We compared the activity of ceftaroline-avibactam (CPA) to that of ertapenem (ERT) against common anaerobic pathogens in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model. Simulations of doses of ceftaroline-fosamil at 600 mg every 8 h (q8h) (maximum free drug concentration [fC_max], 17.04 mg/liter, and half-life [t_1/2], 2.66 h) plus avibactam at 600 mg q8h (fC_max, 11.72 mg/liter, and t_1/2, 1.8 h) and of ertapenem at 1 g q24h (fC_max, 13 mg/liter, and t_1/2, 4 h) were evaluated against two strains of Bacteroides fragilis, one strain of Prevotella bivia, and one strain of Finegoldia magna in an anaerobic one-compartment in vitro PK/PD model over 72 h with a starting inoculum of ∼8 log₁₀ CFU/ml. Bactericidal activity was defined as a reduction of ≥3 log₁₀ CFU/ml from the starting inoculum. Both CPA and ERT were bactericidal against all four strains. CPA demonstrated improved activity against Bacteroides strains compared to that of ERT but had similar activity against Finegoldia magna and P. bivia, although modest regrowth was observed with CPA against P. bivia. No resistance emerged from any of the models. The pharmacokinetics achieved were 92 to 105% of the targets. CPA has potent in vitro activity against common anaerobic pathogens at clinically relevant drug exposures and may be a suitable single product for the management of complicated polymicrobial infections.     

5,6-Dihydro-5-Azathymidine: In Vitro Antiviral Properties Against Human Herpesviruses

5,6-Dihydro-5-azathymidine (DHAdT), a novel water-soluble nucleoside antibiotic, inhibits herpes simplex virus type 1 (HSV-1) in appropriately infected cell cultures to a greater extent than herpes simplex virus type 2 (HSV-2). Vaccinia virus was less susceptible than HSV-2, and pseudorabies virus yields were not reduced at the concentrations studied. Plaque formation by varicella-zoster virus was suppressed by DHAdT. DHAdT was slightly toxic to cells at concentrations that were inhibitory for HSV-1 and varicella-zoster virus. Thymidine and deoxyuridine completely reversed the anti-HSV-1 activity of DHAdT, whereas deoxycytidine was partially effective. Compared with other nucleoside analogs with activity for HSV-1, DHAdT was less active than 5-iodo-2'-deoxyuridine or 1-beta-d-arabinofuranosylcytosine and nearly equipotent with 9-beta-d-arabinofuranosyladenine.     

In Vitro and In Vivo Activities of HPi1, a Selective Antimicrobial against Helicobacter pylori

A high-throughput screen (HTS) was performed to identify molecules specifically active against Helicobacter pylori, the causative agent of peptic ulcer and gastric carcinoma. Currently, treatment of H. pylori infection is suboptimal, with failure rates approaching 25%, despite triple therapy with two broad-spectrum antibiotics and a proton pump inhibitor or quadruple therapy with added bismuth. The HTS was performed in 384-well plates, and reduction of the metabolic indicator resazurin was used as a reporter for cell growth. Diverse molecules from commercial sources were identified as hits, and in vitro validations included measurements of MIC and time-dependent killing as well as anaerobic susceptibility testing against a panel of gut microbes. In vivo validation included testing in the mouse model of H. pylori infection. The small molecule HPi1 (3-hydrazinoquinoxaline-2-thiol) had excellent potency, with an MIC of 0.08 to 0.16 μg/ml and good selectivity for H. pylori compared to a panel of commensal bacteria. HPi1 was also effective in a mouse model of H. pylori infection, reducing colony counts to below the limit of detection after oral dosing of 25 mg/kg/day for 3 days. HPi1 is a promising lead in the search for more effective and specific H. pylori therapeutics.     

In Vitro and In Vivo Inhibition of Murine Gamma Herpesvirus 68 Replication by Selected Antiviral Agents

We have evaluated the susceptibility of the murine gamma herpesvirus 68 (MHV-68) to a variety of antiviral agents. The acyclic nucleoside phosphonate analogs cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine], (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA), and adefovir [9-(2-phosphonylmethoxyethyl)adenine] efficiently inhibited the replication of the virus in Vero cells (50% effective concentrations [EC₅₀s], 0.008, 0.06, and 2.2 μg/ml, respectively). Acyclovir, ganciclovir, and brivudin [(E)-5-(2-bromovinyl)-2′-deoxyuridine] had equipotent activities (EC₅₀s, 1.5 to 8 μg/ml), whereas foscarnet and penciclovir were less effective (EC₅₀s, 23 and ≥30 μg/ml, respectively). The novel N-7-substituted nucleoside analog S2242 [7-(1,3-dihydroxy-2-propoxymethyl)purine] inhibited MHV-68 replication by 50% at 0.2 μg/ml. The susceptibilities of MHV-68 and Epstein-Barr virus (EBV) to cidofovir, HPMPA, adefovir, and acyclovir were found to be comparable. However, for penciclovir, ganciclovir, brivudin, and S2242, major differences in the sensitivity of MHV-68 and EBV were observed, suggesting that MHV-68 is not always an optimal surrogate for the study of antiviral strategies for EBV. When evaluated with a model for lethal MHV-68 infections in mice with severe combined immunodeficiency, cidofovir proved to be very efficient in protecting against virus-induced mortality (100% survival at 50 days postinfection), whereas acyclovir, brivudin, and adefovir had little or no effect.     

In Vitro and In Vivo Antidermatophyte Activities of NND-502, a Novel Optically Active Imidazole Antimycotic Agent

In vitro and in vivo antidermatophyte activities of NND-502, a new imidazole antimycotic agent, were compared with those of two existing antifungal agents, lanoconazole and terbinafine. NND-502 exhibited strong in vitro antifungal activity against Trichophyton spp.; its MIC was 1 to 4 times lower than that of lanoconazole or terbinafine. In an in vivo study with a guinea pig model of tinea pedis, 7-day topical treatment with a 0.5% solution of NND-502 (dissolved in polyethylene glycol 400) was more effective than that with a 0.5% solution of either lanoconazole or terbinafine for eradicating fungi from the infected feet. When the duration of treatment was shortened to 3 days, a topical 1% solution of NND-502 achieved a complete mycological cure, while topical 1% solutions of lanoconazole and terbinafine did not.     

In Vitro and In Vivo Activities of CS-758 (R-120758), a New Triazole Antifungal Agent

The activity of CS-758 (R-120758), a new triazole antifungal agent, was evaluated and compared with those of fluconazole, itraconazole, and amphotericin B in vitro and with those of fluconazole and itraconazole in vivo. CS-758 exhibited potent in vitro activity against clinically important fungi. The activity of CS-758 against Candida spp. was superior to that of fluconazole and comparable or superior to those of itraconazole and amphotericin B. CS-758 retained potent activity against Candida albicans strains with low levels of susceptibility to fluconazole (fluconazole MIC, 4 to 32 microg/ml). Against Aspergillus spp. and Cryptococcus neoformans, the activity of CS-758 was at least fourfold superior to those of the other drugs tested. CS-758 also exhibited potent in vivo activity against murine systemic infections caused by C. albicans, C. neoformans, Aspergillus fumigatus, and Aspergillus flavus. The 50% effective doses against these infections were 0.41 to 5.0 mg/kg of body weight. These results suggest that CS-758 may be useful in the treatment of candidiasis, cryptococcosis, and aspergillosis.     

In Vitro Antiviral Activity of AG7088, a Potent Inhibitor of Human Rhinovirus 3C Protease

AG7088 is a potent, irreversible inhibitor of human rhinovirus (HRV) 3C protease (inactivation rate constant (k(obs)/[I]) = 1,470,000 +/- 440,000 M(-1) s(-1) for HRV 14) that was discovered by protein structure-based drug design methodologies. In H1-HeLa and MRC-5 cell protection assays, AG7088 inhibited the replication of all HRV serotypes (48 of 48) tested with a mean 50% effective concentration (EC(50)) of 0.023 microM (range, 0.003 to 0.081 microM) and a mean EC(90) of 0.082 microM (range, 0.018 to 0.261 microM) as well as that of related picornaviruses including coxsackieviruses A21 and B3, enterovirus 70, and echovirus 11. No significant reductions in the antiviral activity of AG7088 were observed when assays were performed in the presence of alpha(1)-acid glycoprotein or mucin, proteins present in nasal secretions. The 50% cytotoxic concentration of AG7088 was >1,000 microM, yielding a therapeutic index of >12,346 to >333,333. In a single-cycle, time-of-addition assay, AG7088 demonstrated antiviral activity when added up to 6 h after infection. In contrast, a compound targeting viral attachment and/or uncoating was effective only when added at the initiation of virus infection. Direct inhibition of 3C proteolytic activity in infected cells treated with AG7088 was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of radiolabeled proteins, which showed a dose-dependent accumulation of viral precursor polyproteins and reduction of processed protein products. The broad spectrum of antiviral activity of AG7088, combined with its efficacy even when added late in the virus life cycle, highlights the advantages of 3C protease as a target and suggests that AG7088 will be a promising clinical candidate.