U. Wollert

Human serum albumin as a 'silent receptor' for drugs and endogenous substances.

Most drugs are bound to human serum albumin (HSA) via a few high affinity binding sites and several sites of much lower affinity. There is now increasing evidence that the actual number of high affinity drug-binding sites of HSA is rather small. Thus, each of these binding sites binds several drugs of very different chemical and pharmacological properties with relative high affinity. On the other hand these drug-binding sites can in some cases be very specific and even stereoselective, as demonstrated by the stereospecific binding of drugs to some of these binding sites. The discrepancy between both observations can be explained by the conformational adaptability of the HSA-binding sites.

Benzodiazepine antagonism by harmane and other β-carbolines in vitro and in vivo

Abstract Harmane and other related β-carbolines are putative endogenous ligands of the benzodiazepine receptor. Since the compounds are potent convulsants they may have agonist activities at the benzodiazepine receptor while the benzodiazepines may be antagonists. This hypothesis was proved by comparing the in vivo and in vitro antagonism of benzodiazepines by harmane and other β_carboliness. Harmane is clearly a competitive inhibitor of benzodiazepine receptor binding in vitro. Moreover, harmane-induced convulsions can be inhibited reversibly by diazepam in a manner which is consistent with the assumption of competitive antagonism in vivo. For some β-carboline derivatives a correlation was found between the affinity for the benzodiazepine receptor in vitro and the convulsive potency in vivo. Thus, the data reported suggest that harmane or other related β-carbolines are putative endogenous agonists of the benzodiazepine receptor. This suggestion is further supported by the observation that diazepam is equally potent in inhibiting harmane- or picrotoxin-induced convulsions, indicating a convulsive mechanism within the GABA receptor-benzodiazepine receptor system.

1-Methyl-β-carboline (Harmane), a potent endogenous inhibitor of benzodiazepine receptor binding

SummaryThe interaction of several β-carbolines with specific [3H]-flunitrazepam binding to benzodiazepine receptors in rat brain membranes was investigated. Out of the investigated compounds, harmane and norharmane were the most potent inhibitors of specific [3H]-flunitrazepam binding, with IC50-values in the micromolar range. All other derivatives, including harmine, harmaline, and several tetrahydroderivatives were at least ten times less potent. Harmane has been previously found in rat brain and human urine, so it is the most potent endogenous inhibitor of specific [3H]-flunitrazepam binding known so far, with a several hundred fold higher affinity for the benodiazepine receptor than inosine and hypoxanthine. Thus, we suggest that harmane or other related β-carbolines could be potential candidates as endogenous ligands of the benzodiazepine receptor.

On the neuropharmacology of Harmane and other β-carbolines

β-Carbolines have been recently proposed as candidates for the unknown endogenous ligand of the benzodiazepine receptor. Out of the β-carboline derivatives already found in the mammalian CNS, harmane is clearly the most potent inhibitor of benzodiazepine receptor binding. Therefore, it has been considered as possible endogenous ligand for this new receptor system. However, a certain degree of specificity might be a basic condition to accept the hypothesis of harmane as the endogenous ligand. Thus, the effects of harmane as well as other β-carbolines on several neurotransmitter receptor binding systems in vitro and on some neuropharmacological tests in vivo were investigated. Harmane developed the highest affinity towards the benzodiazepine binding site among all systems investigated. Its IC50-values for inhibiting opiate and muscarinic cholinergic receptor binding were about four times lower than those for dopamine and serotonin receptor binding but were about four times higher than that found for the benzodiazepine receptor binding. Norharmane exerted a remarkable displacing activity only at the benzodiazepine binding site. Harmine affected mainly the opiate and cholinergic muscarinic system, whereas tetrahydronorharmane turned out to be a potent inhibitor of serotonin and dopamine receptor binding. Doses of harmane needed to produce convulsions as indication of its possible benzodiazepine receptor agonistic properties are also sufficient to diminish nociception and decrease body temperature whereas the apomorphine-induced licking rate was affected at higher doses. The data demonstrate that harmane affects not only the benzodiazepine binding site but also other neuronal mechanisms. Furthermore, only minor changes of the β-carboline structure lead to substantially different effects. Therefore, the search for other β-carbolines with higher affinity for the benzodiazepine binding site as harmane seems to be promising.

Characterization of the binding of benzodiazepines to human serum albumin.

SummaryThe binding of eleven benzodiazepine derivatives to human serum albumin (HSA) was determined by means of sephadex gel filtration.The albumin binding of the substances was characterized by the percentage of bound drug, the binding constants k+, K1 and m, the number of binding sites per albumin molecule, and the free binding energy. Under the conditions chosen in these experiments there seems to exist only one binding site of the same type for all investigated benzodiazepines at the HSA molecule. The affinities of the benzodiazepines to this binding site are very different. It is discussed which part of the benzodiazepine molecule represents the main binding group.

A highly reactive tyrosine residue as part of the indole and benzodiazepine binding site of human serum albumin

The interaction of L-tryptophan and four benzodiazepine derivatives with tyrosine-modified human serum albumin was investigated by equilibrium dialysis and circular dichroism measurements. Out of the 18 tyrosine residues of the human serum albumin molecule, only 9 could be modified with tetranitromethane. At least up to a degree of modification of 5, the conformation of human serum albumin was not changed and no crosslinking and fractionation has been found, as revealed from circular dichroism measurements in the far ultraviolet range and from SDS polyacrylamide electrophoresis. The modification of only 2 out of the 9 accessible tyrosine residues of human serum albumin strongly affects the binding of L-tryptophan and diazepam to their common, stereospecific bindining site. This was evidently shown by a reduction of the association constants by more than 90% and by a large reduction of the extrinsic Cotton effects of four benzodiazepines bound to human serum albumin. The numbers of binding sites remained unchanged. Strong evidence was presented that only one tyrosine residue, which reacts faster with tetranitromethane than all others, is mainly involved in the specific indole and benzodiazepine binding site of human serum albumin. The location of this highly reactive tyrosine residue and that of the specific indole and benzodiazepine binding site within the human serum albumin primary structure is discussed.

Benzodiazepines: Specific competitors for the binding of l-tryptophan to human serum albumin

SummaryBy means of the gel filtration technique, the effect of nine benzo-diazepine derivates on the binding of l-tryptophan to human serum albumin was investigated. Using equimolar tryptophan and benzodiazepine concentrations, all benzodiazepines with binding constants higher than 104 (M−1), displace l-tryptophan from its binding site to a high degree. The mechanism of the displacement was characterized as a competition for a common binding site. Some of the benzodiazepines displace l-tryptophan to a greater extent than salicylic acid. The benzodiazepines and tryptophan are the only substances known with a high degree of stereospecific binding to human serum albumin. This study shows that there is only one binding site on the human serum albumin molecule, which binds tryptophan and the benzodiazepines in a highly stereospecific manner. Therefore it is concluded that the benzodiazepines and l-tryptophan must have similarities in their molecular structure, so that both can bind to the common binding site in such specific manner. These considerations are discussed in regard to the known influence of benzodiazepine derivatives on the l-tryptophan metabolism in brain. A direct involvement of the reported displacement in the pharmacological actions of the drugs seems not to be relevant because of their small therapeutical plasma levels.

Inhibition of GABA and benzodiazepine receptor binding by penicillins

Penicillins are thought to be GABA receptor antagonists. In order to determine the affinities of various penicillin derivatives for the GABA receptor, their potencies as inhibitors of specific [3H]GABA binding to rat brain membranes were investigated. All investigated penicillins inhibit specific [3H]GABA binding, with IC50 values ranging from 2 to 60 mM. The results are consistent with the assumption that penicillins are weak GABA receptor antagonists.

Influence of pH on the benzodiazepine-human serum albumin complex

SummaryThe influence of pH on the binding of benzodiazepine derivatives to HSA was studied by circular dichroism measurements and by gel filtration.The binding of nearly all benzodiazepines is increased by rising the pH from 6.60 to 8.20. For flurazepam, clonazepam, and nitrazepam this increase in binding is due to an increase of the affinities, while for the other substances the affinity remains constant and the number of binding sites is increased from one to two. The changes in binding of the benzodiazepines by rising the pH are explained by a cationic amino acid residue near or at the benzodiazepine binding site of the HSA molecule. This second binding site is not detectable by circular dichroism. For several of the substances rising the pH from 6.60 to 8.20, is accompanied by large alterations of the optical properties of the HSA-benzodiazepine complexes. These alterations are explained by changes of the asymmetric environment of the benzodiazepine binding site at the HSA molecule in the structural transition at slightly alkaline pH values. To explain the different reactions of the benzodiazepines within the N→B transition a theory is given.

Spectroscopic studies on the complex formation of suramin with bovine and human serum albumin.

The binding of suramin to bovine and human serum albumin was investigated by gel filtration and spectroscopic measurements. Besides some low-affinity binding sites suramin has, on the bovine serum albumin molecule one and on the human serum albumin molecule two, high-affinity binding sites. Spectroscopic measurements reveal that there are large differences between the albumins in the mechanism of binding to the high-affinity binding sites. Further, it is suggested that high concentrations of suramin provoke an unfolding of the albumin moleculse. In order to explain the unusual behaviour of suramin in connection with the displacement of other ligands from the albumin binding the fluorescence probe 1-anilino-8-naphthalenesulfonic acid (ANS) was employed as a reporter group molecule for fluorescence as well as circular dichroism measurements. By these measurements it could be shown that suramin greatly influences the microorganization of both albumin molecules. In the case of these measurements large differences between bovine and human serum albumin were also found.