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Biomedical subjects

A Billich

Publications and source records attributed to A Billich.

At least 37 records · Page 2Linked to original sources
PubMed1996

Paracyclophanes: a novel class of water-soluble inhibitors of HIV proteinase.

A versatile synthesis of functionalized para- and metacyclophanes (macrocycles with one or more aromatic rings incorporated; ansa-compounds) has been developed. Cyclophanes constitute a novel building block for potent human immunodeficiency virus (HIV) protease inhibitors. The synthesis of the macrocyclic ring system was achieved by regio- and stereospecific ring opening of N-protected 4-amino-2,3-epoxy-5-phenylpentanoates with appropriate alpha, omega-diamines and consecutive ring closure under high dilution conditions. The resulting macrocyclic building blocks enabled further broad and flexible derivation. Paracyclophanes, containing oxyethylene substructures, were found to dissolve in phosphate-buffered saline at concentrations as high as 3 mg/mL at physiological pH. Several derivatives with Ki values lower than 10 nM and antiviral activities in the range of 15-50 nM have been obtained. The influence of the ring size and of the substitution pattern of the cyclophane moiety on enzyme inhibition, antiviral activity, and water solubility are discussed. Preliminary data on oral bioavailability in mice are given for selected compounds.

Animals↗
PubMed1996

Inhibitors of human immunodeficiency virus type 1 protease containing 2-aminobenzyl-substituted 4-amino-3-hydroxy-5-phenylpentanoic acid: synthesis, activity, and oral bioavailability.

Systematic modifications of HIV protease inhibitor (2R,3S,4S)-4-[[(benzyloxycarbonyl)-L-valyl]-amino]-3-hydroxy-2-[(4- methoxybenzyl)amino]-5-phenylpentanoyl)-L-valine 2-(aminomethyl)- benzimidazole amide led to a novel series of inhibitors with shortened, modified carboxy terminus. Their synthesis, in vitro enzyme inhibitory data, and antiviral activities are reported. Of particular interest are derivatives featuring the (1S,2R)-1-amino-2-hydroxyindan moiety at the P2'-position since some of them exhibit substantial oral bioavailability in mice. The influence of aqueous solubility and structural parameters on the oral resorption of the inhibitors is discussed. Optimum enhancement of oral bioavailability was observed with L-tert-leucine in P2-position, resulting in the discovery of (2R,3S,4S)-4-[[(benzyloxycarbonyl)-L-tert-leucyl]- amino]-3-hydroxy-2-[(4-methoxybenzyl)amino]-5-phenylpentanoic acid (1S,2R)-1-amino-2-hydroxyindan amide which combines high antiviral activity (IC50 = 250 nM) with a good pharmacokinetic profile (AUC = 82.5 microM.h at a dose of 125 mg/kg po in mice).

Administration, Oral↗
PubMed1995

SDZ PRI 053, an orally bioavailable human immunodeficiency virus type 1 proteinase inhibitor containing the 2-aminobenzylstatine moiety.

A series of inhibitors of human immunodeficiency virus type 1 (HIV-1) proteinase containing the 2-aralkyl-amino-substituted statine moiety as a novel transition-state analog was synthesized, with the aim to obtain compounds which combine anti-HIV potency with oral bioavailability. The reduced-size 2-aminobenzylstatine derivative SDZ PRI 053, which contains 2-(S)-amino-3-(R)-hydroxyindane in place of an amino acid amide, is a potent and orally bioavailable inhibitor of HIV-1 replication. The antiviral activity of SDZ PRI 053 was demonstrated in various cell lines, in primary lymphocytes, and in primary monocytes, against laboratory strains as well as clinical HIV-1 isolates (50% effective dose = 0.028 to 0.15 microM). Cell proliferation was impaired only at 100- to 300-fold-higher concentrations. The mechanism of antiviral action of the proteinase inhibitor SDZ PRI 0.53 was demonstrated to be inhibition of gag precursor protein processing. The finding that the inhibitory potency of SDZ PRI 053 in chronic virus infection, determined by p24 release, was considerably lower than that in de novo infection may be explained by the fact that the virus particles produced in the presence of SDZ PRI 053 are about 50-fold less infectious than those from untreated cultures. Upon intravenous administration, half-lives in blood of 100 and 32 min in mice and rats, respectively, were measured. Oral bioavailability of SDZ PRI 053 in rodents was 20 to 60%, depending on the dose. In mice, rats, and dogs, the inhibitor levels after oral administration remained far above the concentrations needed to efficiently block HIV replication in vitro for a prolonged period. This compound is thus a promising candidate for clinical use in HIV disease.

Administration, Oral↗
PubMed1995

Mode of action of SDZ NIM 811, a nonimmunosuppressive cyclosporin A analog with activity against human immunodeficiency virus type 1 (HIV-1): interference with early and late events in HIV-1 replication.

SDZ NIM 811 is a cyclosporin A analog that is completely devoid of immunosuppressive capacity but exhibits potent and selective anti-human immunodeficiency virus type 1 (HIV-1) activity. The mechanism of action of SDZ NIM 811 is clearly different from those of all other anti-HIV agents described so far. In cell-free assays, it is not an inhibitor of reverse transcriptase, protease, integrase, and it does not interfere with Rev or Tat function. SDZ NIM 811 does not down-regulate CD4 or inhibit fusion between infected and uninfected, CD4-expressing cells. p24 production from chronically HIV-infected cells is not impaired either. To elucidate the mode of action of SDZ NIM 811, we performed DNA PCR analysis in HIV-1 IIIB-infected MT4 cells in one cycle of virus replication. The effects of SDZ NIM 811 on the kinetics of viral DNA synthesis, appearance of two-long terminal repeat circles (2-LTR circles), and integration of DNA were studied. SDZ NIM 811 inhibited 2-LTR circle formation in a concentration-dependent manner, which is indicative of nuclear localization of preintegration complexes. Half-maximal inhibition was achieved at 0.17 microgram/ml; this concentration is close to the 50% inhibitory concentrations (0.01 to 0.2 microgram/ml) for viral growth inhibition. As expected, integration of proviral DNA into cellular DNA was also inhibited by SDZ NIM 811. Analysis of the viral particles produced by SDZ NIM 811-treated, chronically infected cells revealed amounts of capsid proteins, reverse transcriptase activity, and viral RNA comparable to those of the untreated control. However, these particles showed a dose-dependent reduction in infectivity (50% inhibitory concentration of 0.028 microgram/ml) which indicates that the assembly process is also impaired by SDZ NIM 811. Gag proteins are postulated to play a role not only in assembly but also in early steps of viral replication, e.g., nuclear localization of the preintegration complex. Recently, it was reported that HIV-1 Gag protein binds to cyclophilin A, the intracellular receptor for cyclosporin A. Interference with Gag-cyclophilin interaction may be the molecular basis for the antiviral activity of cyclosporin A and its analogs.

Antiviral Agents↗
PubMed1995

Mode of action of SDZ NIM 811, a nonimmunosuppressive cyclosporin A analog with activity against human immunodeficiency virus (HIV) type 1: interference with HIV protein-cyclophilin A interactions.

Cyclosporins, in particular the nonimmunosuppressive derivative SDZ NIM 811, exhibit potent anti-human immunodeficiency virus type 1 (HIV-1) activity in vitro. SDZ NIM 811 interferes at two stages of the viral replication cycle: (i) translocation of the preintegration complex to the nucleus and (ii) production of infectious virus particles. Immunosuppressive activity is not correlated with anti-HIV-1 activity of cyclosporins. However, binding to cyclophilin A, the major cellular receptor protein of cyclosporins, is a prerequisite for HIV inhibition: all structural changes of the cyclosporin A molecule leading to loss of affinity to cyclophilin abolished the antiviral effect. Cyclosporin derivatives did not interact directly with HIV-1 proteins; cyclophilin was the only detectable receptor protein for antivirally active cyclosporins. There is no evidence that inhibition of HIV occurs via a gain of function of cyclophilin in the presence of cyclosporins: the complex of cyclophilin A with SDZ NIM 811 does not bind to calcineurin or to any other viral or cellular proteins under conditions in which calcineurin binding to the cyclophilin A-cyclosporin A complex is easily detectable. Thus, the loss of function caused by binding of cyclosporins to cyclophilin seems to be sufficient for the anti-HIV effect. Cyclophilin A was demonstrated to bind to HIV-1 p24gag, and the formation of complexes was blocked by cyclosporins with 50% inhibitory concentrations of about 0.7 microM. HIV-2 and simian immunodeficiency virus are only weakly or not at all inhibited by cyclosporins. For gag-encoded proteins derived from HIV-1, HIV-2, or simian immunodeficiency virus particles, cyclophilin-binding capacity correlated with sensitivity of the viruses to inhibition by cyclosporins. Cyclophilin A also binds to HIV-1 proteins other than gag-encoded proteins, namely, p17gag, Nef, Vif, and gp120env; the biological significance of these interactions is questionable. We conclude that HIV-1 Gag-cyclophilin A interaction may be essential in HIV-1 replication, and interference with this interaction may be the molecular basis for the antiviral activity of cyclosporins.

Amino Acid Isomerases↗
PubMed1994

Inhibitors of HIV-1 proteinase containing 2-heterosubstituted 4-amino-3-hydroxy-5-phenylpentanoic acid: synthesis, enzyme inhibition, and antiviral activity.

A convenient procedure for the synthesis of 2-heterosubstituted statine derivatives as novel building blocks in HIV-protease inhibitors has been developed. The synthesis starts with protected L-phenylalaninols, which were converted to gamma-amino alpha, beta-unsaturated esters in a one-pot procedure. A highly diastereoselective epoxidation of the N-protected (E)-enoates, followed by regioselective ring opening of the corresponding 2,3-epoxy esters with a variety of heteronucleophiles, resulted in 2-heterosubstituted statine derivatives. The overall stereo-chemical outcome of the transformations meets the required configuration of HIV-protease inhibitors. The short, synthetically flexible, and highly diastereoselective synthesis of 2-heterosubstituted statines has enabled a broad derivation, covering the S3, S2, and S1'-S3' sites of the enzyme. In a series of 46 derivatives, several potent inhibitors were obtained with Ki values as low as 3.4 nM and antiviral activity in the lower nanomolar-range. The structural parameters of the compounds which determine the potency of inhibition and selectivity for the viral enzyme are discussed.

Amino Acid Sequence↗
PubMed1994

In vitro activity of inhibitors of late stages of the replication of HIV in chronically infected macrophages.

Because of the importance of macrophages in the pathogenesis of the disease caused by HIV, we investigated the efficacy of various anti-HIV drugs in human primary macrophages acutely or chronically infected by this virus. The results obtained for acutely infected macrophages show that dideoxynucleosides (AZT, ddI, and ddC), interferon-alpha and -gamma, mismatched double-stranded RNA, Tat inhibitor, phosphorothioate antisense, and inhibitors of HIV protease, all significantly inhibit virus replication at concentrations far below those toxic for the cells. However, in macrophages in which proviral DNA is already integrated (chronically infected macrophages), only the three inhibitors of HIV protease induced significant virus inhibition at concentrations 100 or more times higher than those effective in acutely infected macrophages. Treatment of macrophages with macrophage colony-stimulating factor does not affect the anti-HIV efficacy of protease inhibitors. These results suggest that therapeutic strategies with activity for macrophages, including inhibitors of HIV protease, are worth pursuing in patients with HIV infection.

Cells, Cultured↗
PubMed1994

HIV proteinase inhibitors containing 2-aminobenzylstatine as a novel scissile bond replacement: biochemical and pharmacological characterization.

Derivation of the 2-aminobenzylstatine containing HIV-1 proteinase (PR) inhibitor I led to a series of compounds with considerably improved antiviral activity, the most potent derivatives inhibiting HIV-1 with IC50 values below 25 nM. This was achieved by the combination of several structural modifications, most prominently by introduction of a benzimidazole heterocycle into the inhibitor. The mode of action of the 2-aminobenzylstatine PR inhibitors was demonstrated to be inhibition of gag precursor processing. The antiviral efficacy of the PR inhibitors was demonstrated in various cell lines, in primary T4 lymphocytes and in monocytes. The most potent compound (XI) inhibited replication of several HIV-1 clinical isolates in primary cells with IC50 values of 8 to 23 nM. The analysis of the pharmacokinetic behaviour of compounds I and VII revealed blood half-lives in rodents in the range of about 1.5 h. Compound I also showed appreciable oral uptake in mice (18%), but yielded no detectable blood levels in rats after oral administration. Benzimidazole containing compounds like VII were not orally bioavailable to a significant extent, neither in mice nor in rats. Thus, while introduction of a benzimidazole group into the PR inhibitors was a successful structural modification with regard to antiviral activity in cell culture, it completely abolished oral bioavailability.

Amino Acid Sequence↗
PubMed1994

Inhibition of human immunodeficiency virus type 1 replication by SDZ NIM 811, a nonimmunosuppressive cyclosporine analog.

(Me-Ile-4)cyclosporin (SDZ NIM 811) is a 4-substituted cyclosporin which is devoid of immunosuppressive activity but retains full capacity for binding to cyclophilin and exhibits potent anti-human immunodeficiency virus type 1 (HIV-1) activity. SDZ NIM 811 selectively inhibits HIV-1 replication in T4 lymphocyte cell lines, in a monocytic cell line, and in HeLa T4 cells. Furthermore, its antiviral activity against laboratory strains and against clinical isolates from geographically distinct regions in primary T4 lymphocytes and in primary monocytes (50% inhibitory concentration = 0.011 to 0.057 micrograms/ml) was demonstrated. SDZ NIM 811 does not inhibit proviral gene expression or virus-specific enzyme functions, either free or bound to cyclophilin. The compound does not influence CD4 expression or inhibit fusion between virus-infected and uninfected cells. SDZ NIM 811 was, however, found to block formation of infectious particles from chronically infected cells. Oral administration to mice, rats, dogs, and monkeys resulted in levels in blood considerably exceeding the drug concentration, which completely blocked virus replication in primary cells. SDZ NIM 811 caused changes of toxicity parameters in rats to a smaller degree than cyclosporine (formerly cyclosporin A). Thus, the potent and selective anti-HIV-1 activity of SDZ NIM 811 and its favorable pharmacokinetic behavior together with its lower nephrotoxicity than that of cyclosporine make this compound a promising candidate for development as an anti-HIV drug.

Amino Acid Isomerases↗
PubMed1993

The inhibition of HIV-1 protease by interface peptides.

Previous studies have shown that some peptides derived from one of the terminal amino acid segments of the homodimeric HIV-1 protease show moderate inhibition of this enzyme probably by interfering with the "interface" structure formed by the four terminal segments of the dimer. Different peptides, with improved inhibitory potency, were devised by computer modelling, synthesized, and tested. Ac-TVSFNF, the short peptide with the best inhibition so far (IC50 = 80 microM) is identical with the C-terminal part of the gag-pol frame shift protein p6*. This suggests a regulatory function of p6* as a dimerization inhibitor of HIV protease in the virion. Peptides derived from the active site sequence of PR are inactive. The two terminal hexapeptides of reverse transcriptase are also inactive in the HIV-1 PR activity assay.

Amino Acid Sequence↗
PubMed1992

The N-terminal region of HIV-1 integrase is required for integration activity, but not for DNA-binding.

HIV-1 integrase binds to both double- and single-stranded DNA with Kd-values of around 20 nM, irrespective of sequence similarities with the termini of the viral LTR. For integration activity, however, the correct LTR sequence of the substrate is required. The putative zinc-binding site present at the N-terminus of the protein is not essential for DNA binding, since deletion mutants of the protein lacking this sequence show similar affinity towards DNA as the wild-type; however, these mutants are not capable of performing the LTR-cleavage and integration reactions. Thus, it appears that the N-terminal part of the integrase is essential for catalytic activity.

Base Sequence↗
PubMed1991

Cleavage of phosphorylase kinase and calcium-free calmodulin by HIV-1 protease.

Phosphorylase kinase and calcium-free calmodulin are digested by human immunodeficiency virus-1 protease. In phosphorylase kinase, the alpha subunit is preferentially hydrolyzed at arg748-val749. The beta subunit is cleaved only slowly at leu678-pro679, and calmodulin, the integral delta subunit of phosphorylase kinase, is not cleaved at all. However, free calmodulin in the calcium-depleted form showed to be a good substrate for the protease. Here the cleavage occurs at phe65-pro66 and met71-met72. This fast hydrolysis of free calmodulin can be blocked by micromolar concentrations of Ca2+ or millimolar concentrations of Mg2+.

Amino Acid Sequence↗
PubMed1991

Analysis of subsite preferences of HIV-1 proteinase using MA/CA junction peptides substituted at the P3-P1' positions.

The residues P3, P2, P1, and P1' of a peptide corresponding to the matrix/capsid protein junction in the HIV-1 gag protein (Ser-Gln-Asn-Tyr-Pro-Ile-Val) were systematically replaced and the effect of these single amino acid substitutions on the hydrolysis of each peptide by HIV-1 proteinase was studied. Subsites S1 and S1' of the enzyme showed explicit preference for hydrophobic moieties, but beta-branched amino acids and proline are not tolerated in S1. The S2 subsite shows a preference for small polar and apolar amino acids; it may be occupied by Asn, Asp, Glu, Cys, Ala, or Val, other substitutions, especially by Gln and Ser, prevent hydrolysis of the peptides. In subsite S3 all amino acids except proline can be accommodated.

Amino Acid Sequence↗
PubMed1991

alpha 2-Macroglobulin is cleaved by HIV-1 protease in the bait region but not in the C-terminal inter-domain region.

alpha 2-Macroglobulin is cleaved by human immunodeficiency virus-1 protease. The cleavage site is the Phe684-Tyr685 bond in the "bait region", an exposed part of alpha 2-macroglobulin, creating the "F-form". The methylamine derivative of alpha 2-macroglobulin is also cleaved at the same bond. The homologous chicken ovomacroglobulin does not form an F-form structure with the protease, although, F-form generation by other enzymes is known. This is possibly due to the lack of a suitable cleavage sequence in the corresponding region of ovomacroglobulin. In human alpha 2-macroglobulin, the interdomain segment between the main part of the molecule and the receptor-binding C-terminal domain is not cleaved by the HIV protease although typical cleavage sequences occur. In AIDS, therefore, HIV protease from infected cells in unlikely to interfere with receptor-binding of alpha 2-macroglobulin.

Amino Acid Sequence↗
PubMed1990

Purification, assay and kinetic features of HIV-1 proteinase.

1) The aspartic proteinase of the human immunodeficiency virus type 1 (HIV-1) was purified from cultures of recombinant E. coli. The enzyme preparation is homogeneous as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. 2) A rapid assay procedure for the proteinase was established which makes use of the cleavage of a radiolabeled decapeptide and the separation of substrate and labeled product by ion-exchange resin. 3) Activity of the enzyme is optimal at an ionic strength of 2.5-3.5M; also, the inhibitor pepstatin is a more potent inhibitor at higher ionic strength. This can be attributed to a tighter binding of both substrate and inhibitor in high-salt buffer. 4) The Km value of the decapeptide substrate is independent of the pH in the range of 3.5-7.5, while kcat shows a bell-shaped curve with a maximum at pH 5.2. The shape of the curve can be attributed to pKa values of 4.2 and 6.2 of groups on the enzyme. Pepstatin inhibition is optimal below pH 5.5, but becomes weak above pH 6.

Endopeptidases↗
PubMed1988

Constitutive Expression of Enniatin Synthetase during Fermentative Growth of Fusarium scirpi.

The production of enniatins by Fusarium scirpi during fermentative growth in submerged cultures was measured. The fungus produced the antibiotic during mycelial growth, but not during the stationary phase of cultivation. By contrast, enniatin synthetase, the enzyme responsible for enniatin synthesis, was present during growth, during the stationary phase, and even in spores. Similarly, the enniatin synthetase mRNA was present at every stage of the cultivation of the fungus. Therefore, this multifunctional peptide synthetase is a constitutive enzyme, the expression of which is not regulated by any specific mechanism. The findings stand in contrast to the common assumption that production of secondary metabolites underlies regulatory control, leading to separation of the trophophase and the idiophase.

Journal Article↗