Elin Andersson

Glycine-Amide Is an Active Metabolite of the Antiretroviral Tripeptide Glycyl-Prolyl-Glycine-Amide

ABSTRACT The chemically modified tripeptide glycyl-prolyl-glycine-amide (GPG-NH2) inhibits replication of human immunodeficiency virus (HIV) type 1 (HIV-1) in vitro, probably by interfering with capsid formation. The aim of the present study was to determine whether the metabolites glycyl-proline (GP-OH), glycine (G-OH), prolyl-glycine-amide (PG-NH2), proline (P-OH), and glycine-amide (G-NH2) from proteolytic cleavage may inhibit the replication of HIV-1 in vitro. PG-NH2 has previously been shown to have a modest effect on HIV-1 replication. In the present study we show that G-NH2 exhibits a pronounced inhibitory effect on HIV-1. This effect was not due to a decrease in cell proliferation or viability and could not be shown for herpes simplex virus type 1. The G-NH2 concentration that inhibited virus replication by 50% (IC50) was equimolar to that of GPG-NH2 and ranged from 3 to 41 μM. Transmission electron microscopy revealed that the effect of G-NH2 on HIV-1 morphology was equivalent to that of GPG-NH2 and showed disarranged capsid structures, indicating interference with capsid formation. Serial passage of HIV-infected cells with G-NH2 for more than 20 subcultivations did not decrease the susceptibility to the compound. The results from this study suggest that GPG-NH2 might act as a prodrug and that G-NH2 is an active antiretroviral metabolite.

Type-Specific Human Papillomavirus E6/E7 mRNA Detection by Real-Time PCR Improves Identification of Cervical Neoplasia

ABSTRACT DNA-based human papillomavirus (HPV) assays show high sensitivity but poor specificity in detecting high-grade cervical lesions. Assays detecting mRNA of the oncoproteins E6 and E7 show higher specificity but lack either detection of all high-risk HPV genotypes or the capacity to specify the detected genotypes. Therefore, a real-time PCR assay detecting type-specific E6/E7 mRNA was developed and the clinical performance evaluated. A total of 210 cervical LBC (liquid-based cytology) samples from 204 women were analyzed for HPV DNA and mRNA with the in-house real-time PCR as well as PreTect HPV-Proofer. The sensitivity of real-time PCR mRNA detection to identify histologically confirmed CIN2+ (cervical intraepithelial neoplasia, grade 2 or higher) was 0.91, compared to 0.95 for DNA analysis. The specificity was 0.68 compared to 0.38, and the positive predictive value (PPV) was higher for mRNA (0.67 versus 0.52) without any loss in negative predictive value (NPV). The sensitivity of the real-time PCR mRNA test was somewhat higher than that for PreTect HPV-Proofer (0.83 versus 0.75) in analyses for the same genotypes. The specificities were similar (0.76 versus 0.77). In analyses for mRNA of the eight most common genotypes in cervical cancer (HPV16, -18, -31, -33, -35, -45, -52, and -58), the sensitivity of detection of CIN2+ lesions was 0.87 and the specificity 0.74, with a PPV of 0.70. In conclusion, real-time PCR for detection of HPV E6/E7 mRNA transcripts can be a sensitive and specific tool in screening and investigation of cervical neoplasia. The composition of HPV types in mRNA testing needs to be further investigated to optimize sensitivity and specificity.

Type-dependent E6/E7 mRNA expression of single and multiple high-risk human papillomavirus infections in cervical neoplasia

BACKGROUND Coinfection with multiple HPV types is common in cervical lesions, but the biological significance of the individual infections is difficult to establish. Expression of oncogenic E6/E7 HPV mRNA is correlated to risk of malignant progression, commercial assays for genotyping E6/E7 mRNA of all HR-HPV are lacking. OBJECTIVES To characterize the tendency of 12 HR-HPV to express mRNA, correlated to the severity of the cervical lesion. Furthermore, we wanted to analyse mRNA expression in multiple infections, in order to establish which genotype may be responsible for cellular transformation. STUDY DESIGN 245 samples from women with normal histology, various grades of dysplasia (cervical intraepithelial neoplasia grade 1-3), and cancer, were analysed for presence and genotyping of HPV DNA and mRNA using an in house real-time PCR test. RESULTS Presence of mRNA was detected for 64% of the in total 422 HPV infections present in the samples, and more commonly in high-grade lesions. In 88% of DNA-positive samples from CIN2+ lesions, mRNA could be detected, compared to 33% of DNA-positive samples from women in screening with normal cytology. The genotype most prone to express mRNA in high-grade lesions was HPV45, followed by HPV16 and HPV31, less prone was HPV59. Expression of mRNA was significantly enhanced in CIN2+ lesions, an association also found for HPV16. In 52% of multiple infections (in which mRNA expression was generally more common), more than one genotype expressed mRNA, a phenomenon increasing with severity of lesion. Presence of mRNA could more often be detected in samples with multiple infections than in samples with single infections. CONCLUSIONS The frequent expression of E6/E7 by HPV45 may promote oncogenicity and could be of clinical importance. Since presence of E6/E7 mRNA was common in multiple infections regardless of histology, multiple infection could be a clinically important finding. In multiple HPV infections, mRNA testing may identify the genotype that causes transformation. However, since mRNA expression of several genotypes in one sample is common, further and larger studies using complementary techniques are required.

Activity of the small modified amino acid alpha-hydroxy glycineamide on in vitro and in vivo human immunodeficiency virus type 1 capsid assembly and infectivity

ABSTRACT Upon maturation of the human immunodeficiency virus type 1 (HIV-1) virion, proteolytic cleavage of the Gag precursor protein by the viral protease is followed by morphological changes of the capsid protein p24, which will ultimately transform the virus core from an immature spherical to a mature conical structure. Virion infectivity is critically dependent on the optimal semistability of the capsid cone structure. We have reported earlier that glycineamide (G-NH2), when added to the culture medium of infected cells, inhibits HIV-1 replication and that HIV-1 particles with aberrant core structures were formed. Here we show that it is not G-NH2 itself but a metabolite thereof, α-hydroxy-glycineamide (α-HGA), that is responsible for the antiviral activity. We show that α-HGA inhibits the replication of clinical HIV-1 isolates with acquired resistance to reverse transcriptase and protease inhibitors but has no effect on the replication of any of 10 different RNA and DNA viruses. α-HGA affected the ability of the HIV-1 capsid protein to assemble into tubular or core structures in vitro and in vivo, probably by binding to the hinge region between the N- and C-terminal domains of the HIV-1 capsid protein as indicated by matrix-assisted laser desorption ionization-mass spectrometry results. As an antiviral compound, α-HGA has an unusually simple structure, a pronounced antiviral specificity, and a novel mechanism of antiviral action. As such, it might prove to be a lead compound for a new class of anti-HIV substances.

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.

AlphaHGA; A New Antiviral Substance Against HIV Affecting Capsid Assembly

Background We have previously shown that the tripeptide glycyl-prolyl-glycine-amide (GPG-amide) inhibits HIV-1 replication in vitro by affecting proper capsid assembly of HIV-1. However, GPG-amide failed in a phase II clinical trial on HIV-infected individuals. In the search for what went wrong we have now found that the tripeptide in itself does not exert the antiviral activity but is metabolised in two steps into the active compound by serum enzymes. The first step is cleavage of GPG-amide into GP and glycineamide (G-amide) by the soluble di-peptidyl peptidase CD26. In the present study we show that G-amide is further metabolised to the actve antiviral substance by an enzyme present in foetel bovine serum but not in human serum.