Bicyclams, in which the cyclam (1,4,8,11-tetraazacyclotetradecane) moieties are tethered via an aliphatic bridge (i.e., propylene, as in JM2763) are potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) (E. De Clercq, N. Yamamoto, R. Pauwels, M. Baba, D. Schols, H. Nakashima, J. Balzarini, Z. Debyser, B. A. Murrer, D. Schwartz, D. Thornton, G. Bridger, S. Fricker, G. Henson, M. Abrams, and D. Picker, Proc. Natl. Acad. Sci. USA 89:5286-5290, 1992). We have now found that the bicyclam JM3100, in which the cyclam moieties are tethered by an aromatic bridge [i.e., phenylenebis(methylene)], inhibits the replication of various HIV-1 and HIV-2 strains in various cell lines at a 50% effective concentration (EC50) of 1 to 10 ng/ml, which is about 100-fold lower than the concentration required for JM2763 to inhibit HIV replication and at least 100,000-fold lower than the cytotoxic concentration (> 500 micrograms/ml). In primary T4 lymphocytes or primary monocytes, JM3100 proved inhibitory to HIV-1(IIIB) and several clinical HIV-1 isolates at an EC50 of less than 1 ng/ml. On the basis of time-of-addition experiments, JM3100 appeared to interact with a viral uncoating event, and this was further corroborated by an uncoating assay in which RNase sensitivity of [5-3H]uridine-labeled virions was monitored. In addition, but possibly mechanistically related, JM3100 blocks formation of infectious particles. JM3100 was also found to interfere directly with virus-induced syncytium formation, albeit at a higher concentration (1 to 2 microgram/ml) than that required for inhibition of viral replication. Following subcutaneous injection of 10 mg of JM3100 per kg of body weight to rabbits, anti-HIV activity was detected in serum corresponding to serum drug levels exceeding for at least 6 h by >100-fold the EC(50) required to inhibit HIV replication in vitro. When combined with either 3'-azido-2',3' -dideoxythymidine or 2',3' -dideoxyinosine, JM3100 achieved a additive inhibition of HIV replication, and when repeatedly subcultivated in the presence of JM3100, the virus remained sensitive to the compound for at least 30 passages (120 days) in cell culture.

We have previously described the potent and selective inhibition of several strains of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) by JM2763, an n-propyl-linked dimer of the 1,4,8,11-tetraazamacrocyclic (cyclam) ring system. Upon further investigation, we have also found that incorporating an aromatic rather than aliphatic linker leads to analogs with higher antiviral potency. The prototype, JM3100 (19a, isolated as the octahydrochloride salt), which contains a p-phenylenebis(methylene) moiety linking the cyclam rings, inhibited the replication of HIV-1 (IIIB) and HIV-2 (ROD) at EC50's of 4.2 and 5.9 nM, respectively, while remaining nontoxic to MT-4 cells at concentrations exceeding 421 microM. In order to identify the structural features of bis-tetraazamacrocycles required for potent activity, we have prepared a novel series of phenylenebis(methylene)-linked analogs, in which the macrocyclic ring size was varied from 12 to 16 ring members. Depending upon the substitution of the phenylenebis(methylene) linker (para or meta), sub-micromolar anti-HIV activity was exhibited by analogs bearing macrocycles of 12-14 ring members but with varying cytotoxicity to MT-4 cells. Furthermore, while we found that identical macrocyclic rings are not required for activity, substituting an acyclic polyamine equivalent for one of the cyclam rings in 19a resulted in a substantial reduction in anti-HIV potency, clearly establishing the importance of the constrained macrocyclic structure. A short series of transition metal complexes of 19a were also prepared and evaluated. Complexes of low kinetic stability such as the bis-zinc complex retained activity comparable to that of the parent compound. Finally, the activity of bicyclam analogs appears to be insensitive to the electron-withdrawing or -donating properties of substituents introduced onto the linker, but sterically hindering groups such as phenyl markedly reduced activity. As a result, several analogs with anti-HIV potency comparable to that of 19a have been identified.

No abstract

In vitro evaluation of a large chemical library of pharmacologically acceptable prototpe compounds in a

Although safe and effective vaccines for the prevention of hepatitis B virus (HBV) infection have been available for almost 20 years, the disease remains a major cause of morbidity and mortality worldwide. It is currently estimated that 350 million people are chronic carriers of the virus, often as a result of infection during childhood. Approximately one third to one quarter of these individuals will develop progressive liver disease, including cirrhosis and primary hepatocellular carcinoma (13, 15). Some 1.2 million people die prematurely each year from conditions directly related to HBV infection.Current treatment of chronic hepatitis B aims at interrupting the progression and clinical outcomes of the disease by suppressing viral replication, as evidenced by hepatitis B virus E antigen seroconversion to hepatitis B virus E antibodies (14) or by a decrease in viral load. The first approved therapeutic agent was alpha interferon. Unfortunately, alpha interferon is expensive, is effective in no more than 30% of patients, has to be administered by injection, and is associated with numerous side effects which may necessitate dosage reduction or even treatment discontinuation (13,15).In 1998, the nucleoside analogue lamivudine was approved for use in patients with chronic hepatitis B (7). The convenience of a one-pill-per-day regimen and the low incidence of side effects make it a preferred treatment for many physicians and patients. However, viral breakthrough is detected in approximately 16% to 32% of patients after 1 year of treatment (12). Newer oral nucleoside/nucleotide analogues in clinical trials, such as adefovir dipivoxil (4), entecavir (6), and emtricitabine (19, 18), appear to be at least as potent as lamivudine. In vitro and in vivo studies showed that adefovir and entecavir are also effective in suppressing lamivudine-resistant HBV (8).Mutations in codon 552 [204] (proposed revised nomenclature according to Stuyver et al. [22] is shown in brackets) within the YMDD (tyrosine-methionine-aspartic acid-aspartic acid) motif of the HBV reverse transcriptase/polymerase with substitution of the methionine for valine or isoleucine (M552V/I [M204V/I]) are implicated in the decrease of viral susceptibility to lamivudine (1,5,10,11). In addition, mutations in codons 528 [180] (2, 3, 11) and 555 [207] (9, 17) have also been linked to lamivudine and famciclovir resistance. Sensitive and early detection of emerging resistance may not only help monitor

Background: Total tau (T-tau) and b-amyloid (Ab 1-42 ) levels in cerebrospinal fluid (CSF) can differentiate Alzheimer's disease (AD) from normal aging or depressive pseudo-dementia. Differential diagnosis from dementia with Lewy bodies (DLB) in clinical settings is difficult. Methods: The analytical performance of the INNO-TEST ᮋ PHOSPHO-TAU (181P) assay was validated in terms of selectivity, sensitivity, specificity, precision, robustness, and stability. Clinical utility of the assay alone, or combined with T-tau and Ab 1-42 , for discrimination of AD (ns94) from patients suffering from DLB (ns60) or from age-matched control subjects (CS) (ns60) was assessed in a multicenter study. Results: CSF concentrations of tau phosphorylated at threonine 181 (P-tau 181P ) in AD was significantly higher than in DLB and CS. Discriminant analysis, a classification tree, and logistic regression showed that P-tau 181P was the most statistically significant single variable of the three biomarkers for discrimination between AD and DLB. Conclusions: P-tau 181P quantification is a robust and reliable assay that may be useful in discriminating AD from DLB.

T30177, an oligonucleotide composed of only deoxyguanosine and thymidine, is 17 nucleotides in length and contains single phosphorothioate internucleoside linkages at its 5 and 3 ends for stability. This oligonucleotide does not share significant primary sequence homology with or possess any complementary (antisense) sequence motifs to the human immunodeficiency virus type 1 (HIV-1) genome. T30177 inhibited replication of multiple laboratory strains of HIV-1 in human T-cell lines, peripheral blood lymphocytes, and macrophages. T30177 was also found to be capable of inhibiting multiple clinical isolates of HIV-1 and preventing the cytopathic effect of HIV-1 in primary CD4 ؉ T lymphocytes. In assays with human peripheral blood lymphocytes there was no observable toxicity associated with T30177 at the highest concentration tested (100 M), while the median inhibitory concentration was determined to be in the range of 0.1 to 1.0 M for the clinical isolates tested, resulting in a high therapeutic index for this drug. In temporal studies, the kinetics of addition of T30177 to infected cell cultures indicated that, like the known viral adsorption blocking agents dextran sulfate and Chicago sky blue, T30177 needed to be added to cells during or very soon after viral infection. However, analysis of nucleic acids extracted at 12 h postinfection from cells treated with T30177 at the time of virus infection established the presence of unintegrated viral cDNA, including circular proviral DNA, in the treated cells. In vitro analysis of viral enzymes revealed that T30177 was a potent inhibitor of HIV-1 integrase, reducing enzymatic activity by 50% at concentrations in the range of 0.050 to 0.09 M. T30177 was also able to inhibit viral reverse transcriptase activity; however, the 50% inhibitory value obtained was in the range of 1 to 10 M, depending on the template used in the enzymatic assay. No observable inhibition of viral protease was detected at the highest concentration of T30177 used (10 M). In experiments in which T30177 was removed from infected cell cultures at 4 days post-HIV-1 infection, total suppression of virus production was observed for more than 27 days. PCR analysis of DNA extracted from cells treated in this fashion was unable to detect the presence of viral DNA 11 days after removal of the drug from the infected cell cultures. The ability of T30177 to inhibit both laboratory and clinical isolates of HIV-1 and the experimental data which suggest that T30177 represents a novel class of integrase inhibitors indicate that this compound is a viable candidate for evaluation as a therapeutic agent against HIV-1 in humans.One relatively new approach used in the development of antiviral therapeutics for human immunodeficiency virus type 1 (HIV-1) is the use of oligonucleotides designed as antisense agents (24,26,31). While much effort is being spent on rationally designed oligonucleotides such as antisense agents, there have also been recent findings of multiple alternative mechanisms by which oligonucleotides can i...

Bicyclams are a novel class of antiviral compounds which act as potent and selective inhibitors of the replication of human immunodeficiency virus type 1 (HIV-1) and HIV-2. They block an early step in the viral life cycle following adsorption to the CD4 receptor and preceding reverse transcription. To identify the molecular target of these compounds, we genetically analyzed variants of the HIV-1 molecular clone NL4-3, which developed resistance against two structurally related bicyclams, JM2763 and the more potent SID791. The resistant strains were obtained after long-term passaging in MT-4 cells in the presence of progressively increasing compound concentrations. Recombinants between selected genes of the resistant strains and the parental NL4-3 provirus were generated by adapting the marker rescue technique to MT-4 cells. The bicyclamresistant phenotype was rescued by transferring the envelope gp120 gene of bicyclam-resistant virus into the NL4-3 parental genetic background. In the gp120 genes of the resistant strains, we identified several mutations leading to amino acid substitutions in the V3 loop. Furthermore, two substitutions of highly conserved amino acids in close proximity to the disulfide bridges of the V3 and V4 loops were found in both SID791-and JM2763-resistant strains. Additional mutations in regions encoding V3, C4, V5, and C5 were present in SID791-resistant viruses. Recombination experiments with overlapping parts of the envelope gene indicated that most, if not all, of the mutations were necessary to develop the fully SID791 resistant phenotype. The mutations in the C-terminal part of gp120 downstream of the V3 loop sequence conferred partial resistance to JM2763 but did not significantly decrease susceptibility to SID791. The genetic data and the biological properties of the resistant viruses point to inhibition of entry and fusion as the mode of action of the HIV-inhibitory bicyclams. A possible mechanism of binding of bicyclams to gp120 leading to inhibition of unfolding of gp120 and its shedding from the gp41 fusion domain is discussed.