Selective inhibition of the membrane attack complex of complement by low molecular weight components of the aurin tricarboxylic acid synthetic complex.

Complement plays a vital role in both the innate and adaptive immune systems. It recognizes a target, opsonizes it, generates anaphylatoxins, and directly kills cells through the membrane attack complex (MAC). This final function, which assembles C5b-9(n) on viable cell surfaces, can kill host cells through bystander lysis. Here we identify for the first time compounds that can inhibit bystander lysis while not interfering with the other essential functions of complement. We show that aurin tricarboxylic acid (ATA), aurin quadracarboxylic acid (AQA), and aurin hexacarboxylic acid (AHA), block the addition of C9 to C5b-8 so that the MAC cannot form. These molecules inhibit hemolysis of human, rat, and mouse red cells with a half maximal inhibitory concentration (IC(50)) in the nanomolar range. When given orally to Alzheimer disease type B6SJL-Tg mice, they inhibit MAC formation in serum and improve memory retention. On autopsy, they show no evidence of harm to any organ. Aurin tricarboxylic acid, aurin quadracarboxylic acid, and aurin hexacarboxylic acid may be effective therapeutic agents in Alzheimer disease and other degenerative disorders where self damage from the MAC occurs.

Synthesis and anti-HIV activity of cosalane analogues incorporating two dichlorodisalicylmethane pharmacophore fragments.

A new series of cosalane analogues incorporating two fragments of the dichlorodisalicylmethane pharmacophore has been synthesized. In order to identify the position for the attachment of the pharmacophore fragments to the steroid ring that results in the most potent analogues, two types of compounds were designed. In the first type, the two pharmacophore fragments were attached at C-3 and C-17 of the steroid ring by using appropriate linker units. In the second type, both pharmacophore groups were connected to C-3 of the steroid through an alkenyl chain containing an amide moiety. All of the new compounds displayed antiviral activity versus HIV-1(RF), HIV-1(IIIB), and HIV-2(ROD) in cell culture. The relative potencies of the compounds resulting from the two attachment strategies were found to depend on the viral strain as well as the cell type. Overall, the attachment of the second pharmacophore did not result in either a large gain or a large loss in anti-HIV activity, and the results are therefore consistent with the hypothesis that the two pharmacophores act independently, and one at a time, with positively charged amino acid side chains present on the surface of gp120 and CD4.

Correlation of anti-HIV activity with anion spacing in a series of cosalane analogues with extended polycarboxylate pharmacophores.

Cosalane and its synthetic derivatives inhibit the binding of gp120 to CD4 as well as the fusion of the viral envelope with the cell membrane. The binding of the cosalanes to CD4 is proposed to involve ionic interactions of the negatively charged carboxylates of the ligands with positively charged arginine and lysine amino acid side chains of the protein. To investigate the effect of anion spacing on anti-HIV activity in the cosalane system, a series of cosalane tetracarboxylates was synthesized in which the two proximal and two distal carboxylates are separated by 6--12 atoms. Maximum activity was observed when the proximal and distal carboxylates are separated by 8 atoms. In a series of cosalane amino acid derivatives containing glutamic acid, glycine, aspartic acid, beta-alanine, leucine, and phenylalanine residues, maximum activity was displayed by the di(glutamic acid) analogue. A hypothetical model has been devised for the binding of the cosalane di(glutamic acid) conjugate to CD4. In general, the compounds in this series are more potent against HIV-1(RF) in CEM-SS cells than they are vs HIV-1(IIIB) in MT-4 cells, and they are least potent vs HIV-2(ROD) in MT-4 cells.

Identification of optimal anion spacing for anti-HIV activity in a series of cosalane tetracarboxylates.

The binding of the anti-HIV agent cosalane to CD4 is thought to involve ionic interactions of negatively charged carboxylates of the ligand with positively charged residues on the surface of the protein. An investigation of the optimal anion distances for anti-HIV activity in a series of cosalane tetracarboxylate analogues has been completed, and maximal activity results when the two proximal and the two distal carboxylates are separated by eight atoms.

Synthesis of a cosalane analog with an extended polyanionic pharmacophore conferring enhanced potency as an anti-HIV agent.

A novel cosalane analog having an extended polyanionic pharmacophore was synthesized in order to target specific cationic residues on the surface of CD4. The design rationale is based on a hypothetical binding model of cosalane to the surface of the protein. The new analog displayed an EC50 of 0.55 microM as an inhibitor of the cytopathic effect of HIV-1RF in CEM-SS cells, which represents a significant increase in potency over cosalane itself (EC50 5.1 microM). Both cosalane and the new analog are inhibitors of viral entry into target cells.

Design, synthesis, and biological evaluation of cosalane, a novel anti-HIV agent which inhibits multiple features of virus reproduction.

Cosalane (3), a novel anti-HIV agent having a disalicylmethane unit linked to C-3 of cholestane by a three-carbon linker, was synthesized from commercially available starting materials by a convergent route. Cosalane proved to be a potent inhibitor of HIV with a broad range of activity against a variety of laboratory, drug-resistant, and clinical HIV-1 isolates, HIV-2, and Rauscher murine leukemia virus. The cytotoxicity of cosalane is relatively low as reflected by an in vitro therapeutic index of > 100. Although cosalane inhibits HIV-1 reverse transcriptase and protease, time of addition experiments indicate that it prevents the cytopathic effect of HIV by acting earlier than reverse transcription in the viral replication cycle. The available evidence indicates that the primary mechanism of action of cosalane involves inhibition of gp120-CD4 binding as well as inhibition of a postattachment event prior to reverse transcription.

Preparation and anti-HIV activities of aurintricarboxylic acid fractions and analogues: direct correlation of antiviral potency with molecular weight.

Aurintricarboxylic acid (ATA) was fractionated by a combination of dialysis, ultrafiltration, and gel permeation chromatography. The number average and weight average molecular weights of the ATA fractions were determined by the universal calibration method. The sulfonic acid analogue of ATA was prepared and separated in high and low molecular weight fractions. The phosphonic acid analogue of ATA was also synthesized. All of the ATA fractions were tested for prevention of the cytopathic effect of HIV-1 and HIV-2 in MT-4 cell culture as well as against HIV-1 in CEM cell culture. The abilities of the fractions and analogues to inhibit syncytium formation between HIV-1- and HIV-2-infected HUT-78 cells and uninfected MOLT-4 cells were evaluated. In addition, the fractions and analogues were tested for cytotoxicity in mock-infected MT-4 cells, prevention of the binding of the OKT4A monoclonal antibody to the CD4 receptor, inhibition of the binding of anti-gp120 monoclonal antibody to gp120, inhibition of attachment of HIV-1 virions to MT-4 cells, and inhibition of HIV-1 reverse transcriptase. In all of these assays except cytotoxicity, there was a correlation of potency with molecular weight. The higher the molecular weight, the higher the activity. Several of the lower molecular weight fractions of ATA, which bound to gp120 but not to CD4, prevented HIV-1 and HIV-2 cytopathicity. A similar profile was observed for the phosphonic acid analogue of ATA and the lower molecular weight fraction of the sulfonic acid analogue. The results on the ATA fractions indicate that the binding of ATA to gp120 in the absence of CD4 binding is sufficient for anti-HIV activity. The active compounds bind more avidly to gp120 than to CD4. The anti-HIV activity of the ATA fractions is due to inhibition of virus binding due to an interference with the gp120-CD4 interaction.

Synthesis and anti-HIV activities of low molecular weight aurintricarboxylic acid fragments and related compounds.

Several compounds corresponding to fragments of the schematic representation of the polymeric structure of aurintricarboxylic acid (ATA) have been prepared and tested for prevention of the cytopathic effect of HIV-1 and HIV-2 in MT-4 cell culture and HIV-1 in CEM cell culture. Both the triphenylcarbinol 3 as well as the triphenylmethane 5 were found to afford protection against the cytopathogenicity of HIV-2 in MT-4 cells and HIV-1 in CEM cells, but they were inactive against HIV-1 in MT-4 cells. Both substances were also found to inhibit syncytium formation when MOLT-4 cells were cocultured with HIV-2-infected HUT-78 cells, but were inactive in this assay against HIV-1-infected cells. When observed, the activity is generally moderate in degree of protection and requires concentrations in the 10(-4) molar range. In contrast to ATA, both of these substances were inactive when tested for prevention of the binding of the OKT4A monoclonal antibody to the CD4 receptor and also for inhibition of HIV-1 reverse transcriptase. These substances therefore appear act by a mechanism that is distinct from that of polymeric ATA. Several active and inactive structural analogues of 3 and 5 were also synthesized. The anti-HIV activity in this series seems to depend on the presence of anionic carboxylate groups, since the methyl esters 4, 6, and 12 were uniformly inactive. The diphenylmethanes 8, 14, 18, and 19 also reproducibly inhibited the cytopathic effect of HIV-1 in CEM cell culture.