Investigating hydrophobic ligand-receptor interactions in parathyroid hormone receptor using peptide probes.

With an increasing number of new chemical entities entering clinical studies, and an increasing share of the market, peptides and peptidomimetics constitute one of the most promising classes of therapeutics. The success of synthetic peptides as therapeutics relies on the lead optimization step in which the lead candidates are modified to improve drug-like properties of peptides related to potency, pharmacokinetics, solubility, and stability, among others. Peptidomimetics based on the N-terminal stretch of the first 11 amino acids of the PTH have been investigated as potential lead compounds for the treatment of osteoporosis. On the basis of a peptide reported in the literature, referred to here as the Parent Peptide (H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH2), we conducted systematic SAR analyses to investigate the effects of altering peptide hydrophobicity on PTH receptor functional potency as measured by the cAMP (cyclic adenosine monophosphate) accumulation and ß-arrestin recruitment assays. Among hydrophobic residues, we found that the Val2 position shows the least flexibility in terms of the SAR studies, whereas the Leu7 position appeared to be most flexible. Through circular dichroism and nuclear magnetic resonance spectroscopy studies, we were able to establish that changes in hydrophobic residues significantly change the extent of peptide helicity and that the helical character correlates well with receptor agonist activity. Here, we report several novel PTH 1-11 peptidomimetics that show comparable or enhanced potency to stimulate Gs-signaling over ß-arrestin recruitment as compared with such properties of PTH 1-34 and the Parent Peptide.

Acute murine cytomegalovirus infection: a model for determining antiviral activity against CMV induced hepatitis.

Acute intraperitoneal infection of weanling BALB/c mice with murine cytomegalovirus (MCMV) resulted in an inoculum titer-dependent weight loss, mortality and elevation of plasma transaminases (ALT: alanine transaminase and AST: aspartate transaminase). Three days post infection (p.i.) with 10(4.85) plaque forming units (pfu) there was 90% mortality with a mean death day p.i. of 4.1 +/- 0.2. Plasma levels of ALT and AST were elevated 24- and 15-fold, respectively. Organ titers of virus (log10 pfu/g tissue) were 6.16 in the liver, 6.05 in the spleen, 4.0-4.7 in the lung, heart, kidney and intestine and undetectable in the muscle and brain. Organ concentrations (units/g wet-weight) of ALT were highest in the liver, whilst for AST the highest levels were found in the heart. The concentrations of ALT but not AST were reduced (35-55%) in the infected liver; the concentrations of ALT and AST were not changed in other infected organs. There were excellent correlations (r > 0.95) between viral titers in the liver, increases of plasma ALT and depletion of liver ALT. HPMPC and ganciclovir administered either p.o. or s.c. reduced mortality, increases in plasma transaminases and viral burdens in the liver and prevented depletion of liver ALT. HPMPC was approximately 10-fold more potent than ganciclovir. These results strongly suggest that intraperitoneal infection of the BALB/c mouse with MCMV represents an animal model of CMV hepatitis that can be monitored by measuring plasma ALT.

Dose and duration-dependence of ganciclovir treatment against murine cytomegalovirus infection in severe combined immunodeficient mice.

The present study investigates the full dose-response curve and treatment duration dependence of ganciclovir (GCV) against murine cytomegalovirus (MCMV) infection in severe combined immunodeficiency (SCID) mice. Animals inoculated intraperitoneally with 6.3 x 10(3) pfu of MCMV per mouse developed typical wasting syndrome rapidly and died around day 12 post-inoculation. Once-daily treatment with subcutaneous GCV for 5 days dose dependently delayed MCMV-induced wasting syndrome and mortality at a dose range of 1-80 mg/kg per day, whereas a dose of 160 mg/kg per day induced reversible side-effects. The effect of GCV treatment on mean death day (MDD) was significantly correlated to reductions of viral titers in the lung (r = 0.969, P < 0.05). Treatment duration dependence was examined at the dose of GCV at 80 mg/kg per day for 1, 5, 8 and 12 days. The protective duration, over vehicle-treated mice, was constantly 3-4 days plus the duration of GCV treatment, as evidenced by the delay of viral replication, wasting syndrome and death. At a sub-optimally effective dose of 10 mg/kg per day of GCV, maximum protection was achieved with a 8-day treatment regimen. Prolongation of this treatment to 12 days failed to further delay mean death day and wasting syndrome that started on day 10, indicative of insufficient suppression of viral replication. Treatment with a single dose of GCV failed to show a complete dose-response curve since only minimal protective effects were observed at the dose of 80 mg/kg while side-effects were associated with the dose of 160 mg/kg. The treatment duration dependence and requirement for sufficient dosage of GCV against CMV infection observed in the current model are consistent with clinical observations. It also suggests that 5 8 days treatment duration may be a good balance considering the opportunity for identifying active compounds and speeding up the turnaround time in drug evaluations.

Resistance of herpes simplex virus type 1 to peptidomimetic ribonucleotide reductase inhibitors: selection and characterization of mutant isolates.

Herpes simplex virus (HSV) encodes its own ribonucleotide reductase (RR), which provides the high levels of deoxynucleoside triphosphates required for viral DNA replication in infected cells. HSV RR is composed of two distinct subunits, R1 and R2, whose association is required for enzymatic activity. Peptidomimetic inhibitors that mimic the C-terminal amino acids of R2 inhibit HSV RR by preventing the association of R1 and R2. These compounds are candidate antiviral therapeutic agents. Here we describe the in vitro selection of HSV type 1 KOS variants with three- to ninefold-decreased sensitivity to the RR inhibitor BILD 733. The resistant isolates have growth properties in vitro similar to those of wild-type KOS but are more sensitive to acyclovir, possibly as a consequence of functional impairment of their RRs. A single amino acid substitution in R1 (Ala-1091 to Ser) was associated with threefold resistance to BILD 733, whereas an additional substitution (Pro-1090 to Leu) was required for higher levels of resistance. These mutations were reintroduced into HSV type 1 KOS and shown to be sufficient to confer the resistance phenotype. Studies in vitro with RRs isolated from cells infected with these mutant viruses demonstrated that these RRs bind BILD 733 more weakly than the wild-type enzyme and are also functionally impaired, exhibiting an elevated dissociation constant (Kd) for R1-R2 subunit association and/or reduced activity (kcat). This work provides evidence that the C-terminal end of HSV R1 (residues 1090 and 1091) is involved in R2 binding interactions and demonstrates that resistance to subunit association inhibitors may be associated with compromised activity of the target enzyme.

Regulation of herpes simplex virus true late gene expression: sequences downstream from the US11 TATA box inhibit expression from an unreplicated template.

The true late genes of herpes simplex virus type 1 (HSV-1) are expressed only after the onset of viral DNA replication. Previous studies demonstrated that late promoters lack elements upstream of the TATA box and suggested that only a subset of TATA elements can function in the context of true late promoters. We determined which structural features of true late promoters are responsible for the stringent requirement for viral DNA replication by inserting a series of simple model constructs into the HSV-1 genome in place of one of the two promoters of the UL24 gene. An oligonucleotide consisting of 19 nucleotides spanning the TATA box of the HSV-1 true late US11 gene drove barely detectable levels of expression; by contrast, the corresponding regions of the Adenovirus type 2 major late promoter and the HSV-1 true late glycoprotein C promoter were much more active. Transcripts driven from all of these minimal TATA box promoters accumulated without viral DNA replication. The activity of the US11 TATA box was stimulated by adding upstream Sp1-binding sites or placing the US11 or rabbit beta-globin cap/leader region (-11 to +39) downstream. The Sp1-TATA and TATA-beta-globin cap/leader constructs remained replication independent, while the TATA-US11 cap/leader promoter displayed true late regulation. These results demonstrate that sequences located within the US11 cap/leader region impose a strict requirement for viral DNA replication on a minimal TATA box promoter.

Early abnormal development of calmodulin gene expression and calmodulin-resistant Ca2+-ATPase activity in avian dystrophic muscle.

We have reported previously that the pectoralis muscle from three month-old dystrophic chickens with signs of myopathy exhibits increased calmodulin content, elevated calmodulin-specific mRNA (Biochem. Biophys. Res. Commun. 137:507-512, 1986), and reduced sarcoplasmic reticulum (SR) Ca2+-ATPase activity in response to calmodulin exposure in vitro (Clin. Res. 34: 725A, 1986). To determine the early time sequence for development of these abnormalities, we have studied muscle from embryos and post-hatched chickens at various ages. Quantitated by dot blot analysis, there was an approximate two-fold increase in calmodulin-specific mRNA in dystrophic muscle as early as 13 days ex ovo which was maintained throughout development up to three months ex ovo. Similarly, Ca2+-ATPase activity measured in SR membranes from chickens as early as 13 days post-hatch was also found to be resistant to stimulation in vitro by exogenous calmodulin, whereas the enzyme from normal muscle was calmodulin-stimulable. These findings suggest that the genetic lesion expressed in the avian dystrophic animal model involves the loss of normal control of intracellular calcium metabolism early in the maturation of the affected musculature and prior to appearance of disease signs.

Abnormal expression of the calmodulin gene in muscle from the dystrophic chicken.

Compared to that of genetically-related normal chickens, pectoralis muscle from the dystrophic chicken contained increased calmodulin measured by radioimmunoassay. Determined by the dot blot procedure, expression of the calmodulin gene was enhanced in muscle from affected animals. The bioactivity of the gene product was normal. Together with previous studies reporting increased cell Ca2+ content in dystrophic muscle, the current findings of increased sarcoplasmic calmodulin suggest the latter is a cellular response to defective Ca2+ transport at the level of cell efflux or intracellular organelle (sarcoplasmic reticulum) uptake.

Characterization of lymphocyte interferons with different species specificities from normal and genetically dystrophic chickens.

Lymphocytes from thymus and spleen of normal (Line 412) and genetically dystrophic (Line 413) chickens produce two types of interferons (IFNs) with different host cell specificities. The first type, referred to as ChIFN-alpha, demonstrates antiviral activity on primary normal chicken embryo (CE) cells. This activity is stable at 60 degrees C for 1 h and, in this respect, ChIFN-alpha is similar to the standard ChIFN-beta. In contrast, the second type, referred to as ChIFN-alpha 1, demonstrates antiviral activity in human and simian cells but not in primary CE cells. This activity is labile at 60 degrees C for 1 h. The amount of these two types of IFNs produced in lymphocytes from the spleen of dystrophic chickens was fourfold greater than that produced from normal chickens under similar experimental conditions. In contrast to the lymphocytes from thymus and spleen, the lymphocytes from the bursa of both the normal and dystrophic chickens produced only one type of IFN, namely ChIFN-alpha 1. The development of antiviral state in human cells by ChIFN-alpha 1 requires host RNA synthesis. Although ChIFN-alpha 1 has antiviral properties similar to HuIFN-alpha in human cells, the two IFNs are not antigenically related.