The interleukin-2 and interleukin-4 receptors studied by molecular modelling.

BACKGROUND: Interleukin-2 (IL2) and interleukin-4 (IL4) are members of the four-helix bundle family of cytokines, whose receptors show similarity to each other and to the growth hormone receptor fold. These proteins help to control, among other things, the rate of clonal expansion of lymphocytes, and thus play an important role in the regulation of the immune system. They are therefore of interest as transmembrane signalling proteins, as well as potential pharmaceutical targets. RESULTS: We have modelled structures of the extracellular components of the IL2 and IL4 receptors based on the structure of the complex of human growth hormone with its receptor, and incorporating the recently discovered shared gamma c chain. The models provide possible explanations for several experimental observations, including those from site-directed mutagenesis around the binding sites. Receptor residues that may be close to important side chains on IL2 and IL4 are identified and possible effects of their mutation are discussed. A comparison is made between the models and the growth hormone complex, and between the gamma c chain bound to IL2 and to IL4. CONCLUSIONS: The models offer structural explanations for observed behaviour such as the effects of mutation of the A- and D-helices of the cytokines. In addition, they may be of use in the identification of residues which may interact in the ligand-receptor interfaces, and which would therefore be worthy of further investigation.

Synthesis, structure-activity relationships, and pharmacokinetic properties of dihydroorotate dehydrogenase inhibitors: 2-cyano-3-cyclopropyl-3-hydroxy-N-[3'-methyl-4'-(trifluoromethyl)phenyl ] propenamide and related compounds.

The active metabolite (2) of the novel immunosuppressive agent leflunomide (1) has been shown to inhibit the enzyme dihydroorotate dehydrogenase (DHODH). This enzyme catalyzes the fourth step in de novo pyrimidine biosynthesis. A series of analogues of the active metabolite 2 have been synthesized. Their in vivo biological activity determined in rat and mouse delayed type hypersensitivity has been found to correlate well with their in vitro DHODH potency. The most promising compound (3) has shown activity in rat and mouse collagen (II)-induced arthritis models (ED50 = 2 and 31 mg/kg, respectively) and has shown a shorter half-life in man when compared with leflunomide. Clinical studies in rheumatoid arthritis are in progress.

Stereochemistry of valine biosynthesis. Configuration of the product of rearrangement of alpha-acetolactate.

When alpha-aceto[1,3,5-13C3]lactate (2-hydroxy-2-methyl-3-oxo[1,3,5-13C3]butanoate) was incubated with a cell-free system prepared from Salmonella typhimurium, the valine produced was labelled in the C-4 pro-S position. This result proves that during the tertiary ketol rearrangement catalysed by the reductoisomerase of the isoleucine-valine pathway, the methyl group transfer is to the re face of the trigonal centre at C-3 of alpha-acetolactate.

A computer model of the interleukin-4/receptor complex.

Interleukin-4 is a member of the cytokine family, a group of related messenger proteins which collectively help to moderate and control the immune response. It is believed that the folding topology of the beta-sheets of the interleukin-4 receptor (IL4R) is the same as that seen in the crystal structure of CD4. Although the sequence identity is low, homology modeling techniques have been used to model the IL4R structure from CD4. Refinement by molecular dynamics leads to a suggested structure which has been docked to interleukin-4 (IL4). Several residues of apparent importance for binding are identified.

Analysis of immunoreceptor tyrosine-based activation motif (ITAM) binding to ZAP-70 by surface plasmon resonance.

The signaling function of the T cell antigen receptor (TCR) is mediated via CD3 polypeptides, the cytoplasmic sequences of which bear conserved immunoreceptor tyrosine-based activation motifs (ITAM). ITAM are defined by two YxxL/I sequences separated by a six-eight amino acid long spacer. Upon antigen recognition, ITAM become phosphorylated on both tyrosine residues, creating a high affinity binding site for the tandem SH2 domains found in the protein tyrosine kinase ZAP-70. Using surface plasmon resonance, we further dissected the sequences required for the binding of ZAP-70 to each TCR-associated ITAM. First, we generated protein tyrosine phosphatase-resistant ITAM peptide analogs, in which difluorophosphonomethyl phenylalanyl (F2p) replaced both phosphotyrosines, and showed that those protein tyrosine phosphatase-resistant analogs bind ZAP-70 with high affinity, establishing a rational strategy for the design of novel pharmacological tools capable of interfering with TCR signaling function. Second, we substituted the five amino acids separating the two YxxL/I sequences of the CD3 zeta 1 ITAM with a non-peptidic linker made up of gamma-amino butyric acid units and demonstrated that the length of this intervening sequence rather than its chemical composition is essential for high affinity binding of phosphorylated ITAM to the ZAP-70 SH2 domains.