ABSTRACT
We established a facile access to an unexplored mirror-image library of chiral natural product derivatives using d-protein technology. In this process, two chemical syntheses of mirror-image substances including a target protein and hit compound(s) allow the lead discovery from a virtual mirror-image library without the synthesis of numerous mirror-image compounds.
Subject(s)
Biological Products/chemistry , Biological Products/pharmacology , Drug Evaluation, Preclinical/methods , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Models, Molecular , Protein Conformation , Stereoisomerism , User-Computer InterfaceABSTRACT
Neurokinin B (NKB) regulates the release of gonadotropin-releasing hormone (GnRH) via activation of the neurokinin-3 receptor (NK3R). We evaluated the biological stability of NK3R selective agonists to develop novel NK3R agonists to regulate reproductive functions. On the basis of degradation profiles, several peptidomimetic derivatives were designed. The modification of senktide with (E)-alkene dipeptide isostere generated a novel potent NK3R agonist with high stability and prolonged bioactivity.
Subject(s)
Neurokinin B/analogs & derivatives , Peptide Fragments/agonists , Peptidomimetics/pharmacology , Receptors, Neurokinin-3/agonists , Substance P/analogs & derivatives , Animals , Female , Goats , Humans , Hypothalamus/metabolism , Inhibitory Concentration 50 , Ovariectomy , Peptide Fragments/classification , Peptide Fragments/metabolism , Peptide Hydrolases/metabolism , Peptidomimetics/chemistry , Protein Stability , Serum/metabolism , Substance P/agonists , Substance P/classification , Substance P/metabolism , SwineABSTRACT
The lack of small animal models for the evaluation of anti-human immunodeficiency virus type 1 (HIV-1) agents hampers drug development. Here, we describe the establishment of a simple and rapid evaluation system in a rat model without animal infection facilities. After intraperitoneal administration of test drugs to rats, antiviral activity in the sera was examined by the MAGI assay. Recently developed inhibitors for HIV-1 entry, two CXCR4 antagonists, TF14016 and FC131, and four fusion inhibitors, T-20, T-20EK, SC29EK, and TRI-1144, were evaluated using HIV-1(IIIB) and HIV-1(BaL) as representative CXCR4- and CCR5-tropic HIV-1 strains, respectively. CXCR4 antagonists were shown to only possess anti-HIV-1(IIIB) activity, whereas fusion inhibitors showed both anti-HIV-1(IIIB) and anti-HIV-1(BaL) activities in rat sera. These results indicate that test drugs were successfully processed into the rat sera and could be detected by the MAGI assay. In this system, TRI-1144 showed the most potent and sustained antiviral activity. Sera from animals not administered drugs showed substantial anti-HIV-1 activity, indicating that relatively high dose or activity of the test drugs might be needed. In conclusion, the novel rat system established here, "phenotypic drug evaluation", may be applicable for the evaluation of various antiviral drugs in vivo.
Subject(s)
Anti-HIV Agents/administration & dosage , HIV-1/drug effects , Amino Acid Sequence , Animals , Anti-HIV Agents/blood , Anti-HIV Agents/pharmacokinetics , Biological Availability , Drug Evaluation, Preclinical , Enfuvirtide , HIV Envelope Protein gp41/administration & dosage , HeLa Cells , Humans , Injections, Intraperitoneal , Kinetics , Molecular Sequence Data , Peptide Fragments/administration & dosage , Peptides/administration & dosage , Peptides, Cyclic/administration & dosage , Rats , Receptors, CXCR4/antagonists & inhibitors , Time Factors , Virus Internalization/drug effectsABSTRACT
Entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by its envelope protein gp41 through membrane fusion. Interaction of two extra-virion heptad repeats (HRs) in the gp41 plays a pivotal role in the fusion, and its inhibitor, enfuvirtide (T-20), blocks HIV-1 entry. To identify agents that block HIV-1 fusion, two screening methods based on detection and quantification by the enzyme-linked immunosorbent assay (ELISA) principle have been established. One method uses an alkaline phosphatase (ALP)-conjugated antibody (Ab-ELISA) and the other uses an ALP-fused HR (F-ELISA) to detect and quantify the interaction of the two HRs. The F-ELISA was more simple and rapid, since no ALP-conjugated antibody reaction was required. Both ELISAs detected all the fusion inhibitors tested except for T-20. Interaction of the two HRs was observed in both ELISAs, even in the presence of 10% dimethyl sulfoxide. Ab-ELISA performed best in a pH ranging from 6 to 8, while F-ELISA performed best at a pH ranging from 7 to 8. These results indicate that both established ELISAs are suitable for the identification of HIV-1 fusion inhibitors.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , HIV Envelope Protein gp41/chemistry , HIV Fusion Inhibitors/pharmacology , HIV-1 , Membrane Fusion/drug effects , Repetitive Sequences, Amino Acid/genetics , Alkaline Phosphatase/chemistry , Amino Acid Sequence , Drug Evaluation, Preclinical/methods , HIV Envelope Protein gp41/metabolism , HIV Fusion Inhibitors/chemical synthesis , HIV Fusion Inhibitors/chemistry , HIV-1/drug effects , HIV-1/pathogenicity , Humans , Virion/chemistryABSTRACT
A water-soluble extract of fermented Polygonum tinctorium Aiton (Polygonaceae) called Sukumo, exhibited a potent inhibitory activity against HIV type 1 in vitro. The extract potently suppressed acute HIV-1 (IIIB) infection in MT-4 cells with EC50 values of 0.5 microg/ml but exhibited low cytotoxicity to MT-4 cells even at a high concentration (CC50 > 1000 microg/ml). It also inhibited giant cell formation in co-cultures of HIV-infected cells and uninfected Molt-4 cells. Sukumo extract was found to interact with both the viral envelope glycoprotein and cellular receptors, thus blocking virus-cell binding and virus-induced syncytium formation. There was a good correlation between the extract's anti-HIV-1 activity and its inhibitory effects on HIV-1 binding. It also suppressed replication of herpes simplex virus type 1 in Vero cells with an EC 50 of 11.56 microg/ml. On the other hand, there was no appreciable activity against influenza A virus, poliovirus or SARS corona virus when tested at concentrations ranging from 3.2-400 microg/ml as shown by microscopic image analysis for cytopathic effect (CPE). Physico-chemical studies revealed that the anti-HIV activity in the extract was essentially maintained after boiling at 100 degrees C in 1N HCl or 1N NaOH, and after treatment with 100 mM NaIO4. The inhibitory activity of the extract was also not reduced after pronase digestion. The active factor in the extract is likely to be a novel compound(s) having a polyanionic substructure and a molecular weight of 10,000-50,000.