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We identify three submicromolar inhibitors with new chemical scaffolds for cystathionine γ-lyase (CSE) by a tandem-well-based high-throughput assay. NSC4056, the most potent inhibitor with an IC 50 of 0.6 μM, which is also known as aurintricarboxylic acid, selectively binds to Arg and Tyr residues of CSE active site and preferably inhibits the CSE activity in cells rather than cystathionine β-synthase (CBS), the other H 2 S-generating enzyme. Moreover, NSC4056 effectively rescues hypotension in hemorrhagic shock rats.

Aurintricarboxylic acid (ATA) is a potent inhibitor of many enzymes needed for cell and virus replication, such as polymerases, helicases, nucleases, and topoisomerases. This study examines how ATA interacts with the helicase encoded by the hepatitis C virus (HCV) and reveals that ATA interferes with both nucleic acid and ATP binding to the enzyme. We show that ATA directly binds HCV helicase to prevent the enzyme from interacting with nucleic acids and to modulate the affinity of HCV helicase for ATP, the fuel for helicase action. Amino acid substitutions in the helicase DNA binding cleft or its ATP binding site alter the ability of ATA to disrupt helicase-DNA interactions. These data, along with molecular modeling results, support the notion that an ATA polymer binds between Arg467 and Glu493 to prevent the helicase from binding either ATP or nucleic acids. We also characterize how ATA affects the kinetics of helicase-catalyzed ATP hydrolysis, and thermodynamic parameters describing the direct interaction between HCV helicase and ATA using microcalorimetry. The thermodynamics of ATA binding to HCV helicase reveal that ATA binding does not mimic nucleic acid binding in that ATA binding is driven by a smaller enthalpy change and an increase in entropy.

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.

Several novel alkenyldiarylmethane (ADAM) non-nucleoside HIV-1 reverse transcriptase inhibitors were synthesized. The most potent of these proved to be 3',3"-dibromo-4',4"-dimethoxy-5'5"-bis(methoxycarbonyl)-1,1-diphenyl-1-+ ++heptene (8) ADAM 8 inhibited the cytopathic effect of HIV-1 in CEM cell culture with an EC50 value of 7.1 microM and was active against an array of laboratory strains of HIV-1 in CEM-SS and MT-4 cells, but was inactive as an inhibitor of HIV-2. In common with the other known non-nucleoside reverse transcriptase inhibitors, ADAM 8 was an effective inhibitor of HIV-1 reverse transcriptase (IC50 1 microM) with poly(rC).oligo(dG), but not with poly(rA).oligo(dT), as the template/primer. ADAM 8 was inactive against HIV-1 reverse transcriptases containing non-nucleoside reverse transcriptase inhibitor resistance mutations at residues 101, 106, 108, 139, 181, 188, and 236, while it remained active against enzymes with mutations at residues 74, 98, 100, 103, and at 103/181. An AZT-resistant virus having four mutations in reverse transcriptase was more sensitive to inhibition by ADAM 8 than the wild-type HIV-1. In addition, ADAM 8 displayed synergistic activity with AZT, but lacked synergy with ddI. ADAM 8 or a structurally related analog may therefore be useful as an antiviral agent in combination with AZT or with other NNRTIs that are made ineffective by mutations at residues which do not confer resistance to ADAM 8.

In order to define the role of the cholestane moiety in the anti-HIV agent cosalane, a series of cosalane analogs was synthesized in which the cholestane ring system was replaced by normal alkenyl and phosphodiester substituents having varied chain lengths and lipophilicities. The compounds containing simple alkenyl substituents were found to be more potent as inhibitors of the cytopathic effect of HIV-1 in cell culture than the phosphodiesters. In addition, the potencies of the alkene congeners correlated positively with chain length and lipophilicity of the alkene. The results indicate that the cholestane moiety of cosalane functions as a lipophilic accessory appendage to escort the dichlorodisalicylmethane pharmacophore to a lipid environment.

Several new analogues of the novel anti-HIV agent cosalane have been synthesized and evaluated as inhibitors of HIV-1 integrase and protease, HIV-1 replication, HIV-1 and HIV-2 cytopathicity, HIV-1- and HIV-2-mediated syncytium formation, and cytopathicity of a variety of human pathogenic viruses. The congeners displayed enhanced potencies relative to cosalane itself as inhibitors of HIV-1 integrase and protease. The two most potent analogues against HIV-1 integrase displayed IC50 values of 2.2 microM, while the three most potent compounds against HIV-1 protease had IC50 values in the 0.35-0.39 microM range. In addition to its activity against HIV-1 and HIV-2 cytopathicity, cosalane inhibited the cytopathic effects of herpes simplex virus-1, herpes simplex virus-2, and human cytomegalovirus at concentrations that were well below the cytotoxic concentrations. Potentially useful antiviral activities were also revealed for some of the new cosalane congeners against influenza virus, Junin virus, and Tacaribe virus.

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.

A major event in apoptosis is the digestion of chromatin into oligonucleosomal fragments. However, the enzymes responsible for the DNA degradation have not been well characterized. Here we report the purification of an endonuclease from human spleen cell nuclei that is likely to be responsible for DNA digestion in apoptosis. Enzyme activity was measured by a sensitive fluorometric assay, which assesses the conversion of plasmid DNA from a supercoiled to an open form. The endonuclease was extracted from isolated nuclei with NaCl between 100 and 350 mM and was further purified by chromatography on columns of phosphocellulose, Superdex 75, and chelating Sepharose (Zn2+ form). By gel filtration, the apparent molecular mass was 22-26 kDa; on SDS-polyacrylamide gel electrophoresis, the purified enzyme showed a single 27-kDa band. The enzyme required both Mg2+ (optimum, 5 mM) and Ca2+ (optimum, 2 mM) for activity. It was inhibited by Zn2+ (100% inhibition at 50 microM) and by high (> 10 mM) concentrations of Ca2+. Aurintricarboxylic acid, spermine, p-(hydroxymercuri)benzoate, and N-ethylmaleimide were also endonuclease inhibitors. No inhibition was observed with iodoacetamide, G-actin, or nucleoside 3',5'-bisphosphates. An optimum pH of 8.0 was found. When added to human CCRF-CEM lymphoblast nuclei, that do not contain the endonuclease, the purified splenic enzyme digested the chromatin into the mono- and oligonucleosomal fragments that are characteristic of apoptosis. On the basis of this result, and the observation that the activators and inhibitors of the purified endonuclease closely parallel those that affect apoptosis, it seems likely that this enzyme is involved in the apoptotic degradation of DNA in human lymphocytes.

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.

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.

The Bacillus subtilis RNA polymerase sigma subunit and the phage SPO1-coded gene 28 protein are responsible for selective binding of RNA polymerase to early and middle SPO1 promoters, respectively. The association of the RNA polymerase core with each of these subunits weakens during the elongation of RNA chains. Similar changes are known to be an essential part of the Escherichia coli RNA polymerase sigma cycle.

An unusual type of posttranslational modification has been observed in a rat brain in vitro system. It consists in leucine addition to a preformed protein in such a way that the added leucine is not located at either the NH2 or the COOH terminus of the acceptor protein. The incorporation reaction requires ATP, ATP-generating components and tRNA. It is inhibited by aurintricarboxylic acid but does not require the presence of ribosomes or GTP. The incorporated leucine has a free NH2 group, and it is not released by leucine aminopeptidase or carboxypeptidase A. It is linked to the acceptor protein through a bond that is too alkali labile and too hydroxylamine labile to be a peptide bond. The simplest interpretation of the results consists in proposing that an ester bond is formed between the leucine and the side chain of a serine, threonine, or tyrosine in the acceptor protein.

The mechanism of inhibition of protein--nucleic acid complex formation by polymeric aurintricarboxylic acid (ATA) was investigated by proton magnetic resonance spectroscopy. The approach was the synthesis of totally deuterated ATA, followed by a 100-MHz proton magnetic resonance study of its interaction with bovine pancreatic ribonuclease A (RNase), a model nucleic acid binding protein. The binding of ATA to RNase elicited chemical shift changes and line broadening in the C(2)--H resonances of histidyl residues 12 and 119, both of which are located in the active site, whereas that of histidyl residue 105, which resides on the exterior of the protein structure, is unaffected. (Histidyl residue 48 is not observed under our conditions except at high pH.) The epsilon-methylene protons of the lysyl side chains were also broadened upon the binding of ATA. Polymeric ATA displaces cytidine 2'-monophosphate and cytidine 3'-monophosphate from the active site of the enzyme as revealed by nuclear magnetic resonance spectroscopy. These observations suggest that the mechanism of action of ATA involves competition between the nucleic acid and the polymeric ATA for binding in the active site of the protein. Electron spin resonance spectroscopy reveals that polymeric ATA is a stable free radical, thus accounting for the major line broadening effect upon binding to protein. This finding may provide a powerful means of probing the nucleic acid binding site of proteins by proton magnetic resonance spectroscopy.