A mutational analysis of receptor binding sites of interleukin-1 beta: differences in binding of human interleukin-1 beta muteins to human and mouse receptors.

The 3-D crystal structure of interleukin-1 beta (IL-1 beta) has been used to define its receptor binding surface by mutational analysis. The surface of IL-1 beta was probed by site-directed mutagenesis. A total of 27 different IL-1 beta muteins were constructed, purified and analyzed. Receptor binding measurements on mouse and human cell lines were performed to identify receptor affinities. IL-1 beta muteins with modified receptor affinity were evaluated for structural integrity by CD spectroscopy or X-ray crystallography. Changes in six surface loops, as well as in the C- and N-termini, yielded muteins with lower binding affinities. Two muteins with intact binding affinities showed 10- to 100-fold reduced biological activity. The surface region involved in receptor binding constitutes a discontinuous area of approximately 1000 A2 formed by discontinuous polypeptide chain stretches. Based on these results, a subdivision into two distinct local areas is proposed. Differences in receptor binding affinities for human and mouse receptors have been observed for some muteins, but not for wild-type IL-1 beta. This is the first time a difference in binding affinity of IL-1 beta muteins to human and mouse receptors has been demonstrated.

Receptor binding and biological activity of IL-1 alpha, IL-1 beta, IL-1 beta analogues and an IL-1 antagonist in A375 human melanoma cells.

A receptor binding assay for IL-1 peptides on human melanoma cells of the A 375 cell line is reported. Strains differing in their sensitivity to the cytotoxic effects of IL-1 beta were compared. In both strains, binding equilibrium at temperatures between 0 degrees and 37 degrees C was reached after 4 to 8 hours. At 37 degrees C, most of the bound ligand was rapidly internalized leaving a constant level of surface receptors. Scatchard analysis at 0 degrees C revealed a single class of high affinity receptors with a similar KD in both IL-1 resistant (0.18 +/- 0.07 nM) and sensitive strains (0.14 +/- 0.06 nM) but a 10-fold difference in the number of binding sites. Whereas > 1000 binding sites per cell were regularly observed in all resistant strains, only 100-200 sites could be detected on the IL-1 sensitive cells. In displacement assays, IL-1 beta was found to be slightly more potent than IL-1 alpha in both strains. In an attempt to further characterize the IL-1 binding site in these cells, the binding characteristics and biological activity of 20 point mutations of IL-1 beta were examined. EC50 values similar to those of the wild type peptide were found in all these analogues with the exception R11S and E128K: their EC50 was increased by a factor of 10 but the biological activity was reduced 1000-fold as compared to IL-1 beta. The relative potency of an IL-1 receptor antagonist was similar to that of IL-1 beta in the displacement binding assay but a 100-fold higher concentration was required to completely block the cytotoxic effects of IL-1 beta. These results show that A375 human melanoma cells are useful for screening the binding and biological properties of analogues of the IL-1 family of peptides.

Mapping the receptor binding domain of interleukin-1 beta by means of binding studies using overlapping sequence fragments: why did it fail?

Interleukin-1 (IL-1) alpha and beta are polypeptide hormones that mediate a broad range of biological activities and interact with surface receptors on numerous cell types. Great efforts are made at present to define the interaction domain of IL-1 with its receptor. We have tried to map the domain of IL-1 beta by assessing the receptor interaction of synthetic octapeptide acid amides representing overlapping segments of the IL-1 beta primary sequence. Since the tertiary structure of IL-1 beta is known, the selection of octapeptides could be confined to the surface exposed residues. More than a 100 octapeptides were tested for binding in a competitive binding assay, using a mouse thymoma cell line (EL 4.61) as a receptor source and 125I-IL-1 alpha and beta as radioligands. No binding was found at up to a one hundred fold excess of octapeptide over radioligand. From this lack of binding we conclude that the entropic cost of conformationally freezing the octapeptide is high and that the conformation of the binding domain is per se in terms of free energy and is stabilized by the overall tertiary structure of IL-1.

Regression of "hormone-independent" DMBA-induced mammary carcinomas in response to GP 48989 and its effects on hormone receptors.

GP 48 989 causes regression of DMBA-induced mammary carcinomas of spayed ("hormone-independent"), non spayed (approximately 5% "hormone-dependent") rats and of tumors which had become refractory to estradiol treatment. Since no binding to the cytoplasmic estradiol receptor after prolonged intramuscular treatment was found, the compound does not act as a classical anti-estrogen.

PIP: The effect of GP 48989 (5-methyl-3-(2-methylallyl)-2- ((3-methyl-4-oxo-2-thiazolidinylidene)hydrazono)-4- thiazolidinone) on the regression of "hormone-independent" DMBA-induced mammary tumors in rats and on estradiol receptors was studied. Treatment of carcinomas in spayed (hormone-independent), nonspayed (hormone-dependent) rats, and of tumors which were refractory to treatment with estradiol dipropionate caused regression of the tumors. No bin to the cytoplasmic estradiol receptor was observed after prolonged intramuscular treatment, which indicates that GP 48989 does not act as a typical estrogen antagonist.

Synergistic effect of rifamycin derivatives and amphotericin B on viral transformation of a murine cell line.

One of the most potent inhibitors of RNA-dependent DNA polymerase activity so far described (rifazacyclo-16) was not correspondingly as active in focus inhibition. This discrepancy was thought to be due to the inability of the drug to penetrate the cell membrane. It has been found that a very low level of amphotericin B allows this drug, as well as the previously described 2',6'-dimethyl-N(4')benzyl-N(4')-[desmethyl] rifampicin, to exhibit a very high capability to inhibit focus formation. Since these two drugs are highly lipophilie, their activity may be expected to be dependent upon any lipophilic components in the medium, such as serum or detergents. The use of amphotericin B as well as serum in tissue cultures is common, and could account for some of the variability in focus inhibition reported in the literature.

Detergent effects on a reverse transcriptase activity and on inhibition by rifamycin derivatives.

A reverse transcriptase activity, extracted from virus-transformed cells, is activated by very low concentrations of nonionic detergents. These same detergents also significantly reduce the effectiveness of certain rifamycin derivatives as inhibitors of the polymerase activity when the detergents are present at micelle-forming concentrations.

Effect of rifampicin and two of its derivatives on cells infected with Moloney sarcoma virus.

It is shown that rifampicin, and especially the related antibiotic 2',6'-dimethyl-N(4')-benzyl-N(4')- [desmethyl]rifampicin (DMB-rifampicin) can inhibit focus formation by Moloney sarcoma virus on BALB/3T3 tissue cultures. At 10 mug/ml DMB-rifampicin totally inhibits focus formation while reducing virus replication by at least a factor of fifty and cell proliferation by only a factor of three. These observations, taken together with those of others, suggest a role for an RNA-dependent DNA polymerase and the gene for its synthesis both in normal cell processes and in the transformation process.