Radiosensitization of gliomas by intracellular generation of 5-fluorouracil potentiates prodrug activator gene therapy with a retroviral replicating vector.

A tumor-selective non-lytic retroviral replicating vector (RRV), Toca 511, and an extended-release formulation of 5-fluorocytosine (5-FC), Toca FC, are currently being evaluated in clinical trials in patients with recurrent high-grade glioma (NCT01156584, NCT01470794 and NCT01985256). Tumor-selective propagation of this RRV enables highly efficient transduction of glioma cells with cytosine deaminase (CD), which serves as a prodrug activator for conversion of the anti-fungal prodrug 5-FC to the anti-cancer drug 5-fluorouracil (5-FU) directly within the infected cells. We investigated whether, in addition to its direct cytotoxic effects, 5-FU generated intracellularly by RRV-mediated CD/5-FC prodrug activator gene therapy could also act as a radiosensitizing agent. Efficient transduction by RRV and expression of CD were confirmed in the highly aggressive, radioresistant human glioblastoma cell line U87EGFRvIII and its parental cell line U87MG (U87). RRV-transduced cells showed significant radiosensitization even after transient exposure to 5-FC. This was confirmed both in vitro by a clonogenic colony survival assay and in vivo by bioluminescence imaging analysis. These results provide a convincing rationale for development of tumor-targeted radiosensitization strategies utilizing the tumor-selective replicative capability of RRV, and incorporation of radiation therapy into future clinical trials evaluating Toca 511 and Toca FC in brain tumor patients

Oxpholipin 11D: An Anti-Inflammatory Peptide That Binds Cholesterol and Oxidized Phospholipids

Background: Many Gram-positive bacteria produce pore-forming exotoxins that contain a highly conserved, 12-residue domain (ECTGLAWEWWRT) that binds cholesterol. This domain is usually flanked N-terminally by arginine and C-terminally by valine. We used this 14-residue sequence as a template to create a small library of peptides that bind cholesterol and other lipids. Methodology/Results: Several of these peptides manifested anti-inflammatory properties in a predictive in vitro monocyte chemotactic assay, and some also diminished the pro-inflammatory effects of low-density lipoprotein in apoE-deficient mice. The most potent analog, Oxpholipin-11D (OxP-11D), contained D-amino acids exclusively and was identical to the 14residue design template except that diphenylalanine replaced cysteine-3. In surface plasmon resonance binding studies, OxP-11D bound oxidized (phospho)lipids and sterols in much the same manner as D-4F, a widely studied cardioprotective apoA-I-mimetic peptide with anti-inflammatory properties. In contrast to D-4F, which adopts a stable a-helical structure in solution, the OxP-11D structure was flexible and contained multiple turn-like features. Conclusion: Given the substantial evidence that oxidized phospholipids are pro-inflammatory in vivo, OxP-11D and othe

A Compensatory Mutation Provides Resistance to Disparate HIV Fusion Inhibitor Peptides and Enhances Membrane Fusion

Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide, are peptides designed to competitively inhibit the viral fusion protein gp41. With the emergence of drug resistance, there is an increased need for effective and unique alternatives within this class of antivirals. One such alternative is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide due to its specific antiviral activity, lack of toxicity to cells and tissues, and safety in animals. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. In previous studies, we identified a mutant, R5-tropic virus that had evolved partial resistance to RC-101 during in vitro selection. Here, we report that a secondary mutation in gp41 was found to restore replicative fitness, membrane fusion, and the rate of viral entry, which were compromised by an initial mutation providing partial RC-101 resistance. Interestingly, we show that RC-101 is effective against two enfuvirtide-resistant mutants, demonstrating the clinical importance of RC-101 as a unique fusion inhibitor. These findings both expand our understanding of HIV drug-resistance to diverse peptide fusion inhibitors and emphasize the significance of compensatory gp41 mutations. © 2013 Wood et al

Grifonin-1: A Small HIV-1 Entry Inhibitor Derived from the Algal Lectin, Griffithsin

Background: Griffithsin, a 121-residue protein isolated from a red algal Griffithsia sp., binds high mannose N-linked glycans of virus surface glycoproteins with extremely high affinity, a property that allows it to prevent the entry of primary isolates and laboratory strains of T- and M-tropic HIV-1. We used the sequence of a portion of griffithsin's sequence as a design template to create smaller peptides with antiviral and carbohydrate-binding properties. Methodology/Results: The new peptides derived from a trio of homologous β-sheet repeats that comprise the motifs responsible for its biological activity. Our most active antiviral peptide, grifonin-1 (GRFN-1), had an EC50 of 190.8±11.0 nM in in vitro TZM-bl assays and an EC50 of 546.6±66.1 nM in p24gag antigen release assays. GRFN-1 showed considerable structural plasticity, assuming different conformations in solvents that differed in polarity and hydrophobicity. Higher concentrations of GRFN-1 formed oligomers, based on intermolecular β-sheet interactions. Like its parent protein, GRFN-1 bound viral glycoproteins gp41 and gp120 via the N-linked glycans on their surface. Conclusion: Its substantial antiviral activity and low toxicity in vitro suggest that GRFN-1 and/or its derivatives may have therapeutic potential as topical and/or systemic agents directed against HIV-1

Multivalent Tryptophan‐ and Tyrosine‐Containing [60]Fullerene Hexa‐Adducts as Dual HIV and Enterovirus A71 Entry Inhibitors

Unprecedented 3D hexa-adducts of [60]fullerene peripherally decorated with twelve tryptophan (Trp) or tyrosine (Tyr) residues have been synthesized. Studies on the antiviral activity of these novel compounds against HIV and EV71 reveal that they are much more potent against HIV and equally active against EV71 than the previously described dendrimer prototypes AL-385 and AL-463, which possess the same number of Trp/Tyr residues on the periphery but attached to a smaller and more flexible pentaerythritol core. These results demonstrate the relevance of the globular 3D presentation of the peripheral groups (Trp/Tyr) as well as the length of the spacer connecting them to the central core to interact with the viral envelopes, particularly in the case of HIV, and support the hypothesis that [60]fullerene can be an alternative and attractive biocompatible carbon-based scaffold for this type of highly symmetrical dendrimers. In addition, the functionalized fullerenes here described, which display twelve peripheral negatively charged indole moieties on their globular surface, define a new and versatile class of compounds with a promising potential in biomedical applications

Oxpholipin 11D: an anti-inflammatory peptide that binds cholesterol and oxidized phospholipids.

BackgroundMany gram-positive bacteria produce pore-forming exotoxins that contain a highly conserved, 12-residue domain (ECTGLAWEWWRT) that binds cholesterol. This domain is usually flanked N-terminally by arginine and C-terminally by valine. We used this 14-residue sequence as a template to create a small library of peptides that bind cholesterol and other lipids.Methodology/resultsSeveral of these peptides manifested anti-inflammatory properties in a predictive in vitro monocyte chemotactic assay, and some also diminished the pro-inflammatory effects of low-density lipoprotein in apoE-deficient mice. The most potent analog, Oxpholipin-11D (OxP-11D), contained D-amino acids exclusively and was identical to the 14-residue design template except that diphenylalanine replaced cysteine-3. In surface plasmon resonance binding studies, OxP-11D bound oxidized (phospho)lipids and sterols in much the same manner as D-4F, a widely studied cardioprotective apoA-I-mimetic peptide with anti-inflammatory properties. In contrast to D-4F, which adopts a stable alpha-helical structure in solution, the OxP-11D structure was flexible and contained multiple turn-like features.ConclusionGiven the substantial evidence that oxidized phospholipids are pro-inflammatory in vivo, OxP-11D and other Oxpholipins may have therapeutic potential

Small lipidated anti-obesity compounds derived from neuromedin U

A small library of truncated/lipid-conjugated neuromedin U (NmU) analogs was synthesized and tested in vitro using an intracellular calcium signaling assay. The selected, most active analogs were then tested in vivo, and showed potent anorexigenic effects in a diet-induced obese (DIO) mouse model. The most promising compound, NM4-C16 was effective in a once-weekly-dose regimen. Collectively, our findings suggest that short, lipidated analogs of NmU are suitable leads for the development of novel anti-obesity therapeutics

Classes of Drugs that Mitigate Radiation Syndromes.

We previously reported several vignettes on types and classes of drugs able to mitigate acute and, in at least one case, late radiation syndromes in mice. Most of these had emerged from high throughput screening (HTS) of bioactive and chemical drug libraries using ionizing radiation-induced lymphocytic apoptosis as a readout. Here we report the full analysis of the HTS screen of libraries with 85,000 small molecule chemicals that identified 220 "hits." Most of these hits could be allocated by maximal common substructure analysis to one of 11 clusters each containing at least three active compounds. Further screening validated 23 compounds as being most active; 15 of these were cherry-picked based on drug availability and tested for their ability to mitigate acute hematopoietic radiation syndrome (H-ARS) in mice. Of these, five bore a 4-nitrophenylsulfonamide motif while 4 had a quinoline scaffold. All but two of the 15 significantly (p < 0.05) mitigated H-ARS in mice. We had previously reported that the lead 4-(nitrophenylsulfonyl)-4-phenylpiperazine compound (NPSP512), was active in mitigating multiple acute and late radiation syndromes in mice of more than one sex and strain. Unfortunately, the formulation of this drug had to be changed for regulatory reasons and we report here on the synthesis and testing of active analogs of NPSP512 (QS1 and 52A1) that have increased solubility in water and in vivo bioavailability while retaining mitigator activity against H-ARS (p < 0.0001) and other radiation syndromes. The lead quinoline 057 was also active in multiple murine models of radiation damage. Taken together, HTS of a total of 150,000 bioactive or chemical substances, combined with maximal common substructure analysis has resulted in the discovery of diverse groups of compounds that can mitigate H-ARS and at least some of which can mitigate multiple radiation syndromes when given starting 24 h after exposure. We discuss what is known about how these agents might work, and the importance of formulation and bioavailability

Classes of Drugs that Mitigate Radiation Syndromes.

We previously reported several vignettes on types and classes of drugs able to mitigate acute and, in at least one case, late radiation syndromes in mice. Most of these had emerged from high throughput screening (HTS) of bioactive and chemical drug libraries using ionizing radiation-induced lymphocytic apoptosis as a readout. Here we report the full analysis of the HTS screen of libraries with 85,000 small molecule chemicals that identified 220 "hits." Most of these hits could be allocated by maximal common substructure analysis to one of 11 clusters each containing at least three active compounds. Further screening validated 23 compounds as being most active; 15 of these were cherry-picked based on drug availability and tested for their ability to mitigate acute hematopoietic radiation syndrome (H-ARS) in mice. Of these, five bore a 4-nitrophenylsulfonamide motif while 4 had a quinoline scaffold. All but two of the 15 significantly (p < 0.05) mitigated H-ARS in mice. We had previously reported that the lead 4-(nitrophenylsulfonyl)-4-phenylpiperazine compound (NPSP512), was active in mitigating multiple acute and late radiation syndromes in mice of more than one sex and strain. Unfortunately, the formulation of this drug had to be changed for regulatory reasons and we report here on the synthesis and testing of active analogs of NPSP512 (QS1 and 52A1) that have increased solubility in water and in vivo bioavailability while retaining mitigator activity against H-ARS (p < 0.0001) and other radiation syndromes. The lead quinoline 057 was also active in multiple murine models of radiation damage. Taken together, HTS of a total of 150,000 bioactive or chemical substances, combined with maximal common substructure analysis has resulted in the discovery of diverse groups of compounds that can mitigate H-ARS and at least some of which can mitigate multiple radiation syndromes when given starting 24 h after exposure. We discuss what is known about how these agents might work, and the importance of formulation and bioavailability