A synthetic IgG-binding domain based on staphylococcal protein A was designed with the aid of sequence comparisons and computer graphic analysis. A strategy, utilizing non-palindromic restriction sites, was used to overcome the difficulties of introducing site-specific changes into the repetitive gene. A single mutagenized gene fragment was polymerized to different multiplicities, and the different gene products were expressed in Escherichia coli. Using this scheme, protein A-like proteins composed of different numbers of IgG-binding domains were produced. These domains were changed to lack asparagine--glycine dipeptide sequences as well as methionine residues and are thus, in contrast to native protein A, resistant to treatment with hydroxylamine and cyanogen bromide.

TP53, a critical tumour suppressor gene, is mutated in over half of all cancers resulting in mutant-p53 protein accumulation and poor patient survival. Therapeutic strategies to target mutant-p53 cancers are urgently needed. We show that accumulated mutant-p53 protein suppresses the expression of SLC7A11, a component of the cystine/glutamate antiporter, system xC−, through binding to the master antioxidant transcription factor NRF2. This diminishes glutathione synthesis, rendering mutant-p53 tumours susceptible to oxidative damage. System xC− inhibitors specifically exploit this vulnerability to preferentially kill cancer cells with stabilized mutant-p53 protein. Moreover, we demonstrate that SLC7A11 expression is a novel and robust predictive biomarker for APR-246, a first-in-class mutant-p53 reactivator that also binds and depletes glutathione in tumours, triggering lipid peroxidative cell death. Importantly, system xC− antagonism strongly synergizes with APR-246 to induce apoptosis in mutant-p53 tumours. We propose a new paradigm for targeting cancers that accumulate mutant-p53 protein by inhibiting the SLC7A11–glutathione axis.

Protein engineering techniques were used to construct a derivative of the serine protease subtilisin that ligates peptides efficiently in water. The subtilisin double mutant in which the catalytic Ser221 was converted to Cys (S221C) and Pro225 converted to Ala (P225A) has 10-fold higher peptide ligase activity and at least 100-fold lower amidase activity than the singly mutated thiolsubtilisin (S221C) that was previously shown to have some peptide ligase activity [Nakatsuka, T., Sasaki, T., & Kaiser, E. T. (1987) J. Am. Chem. SOC. 109, 3808-38101. A 1.5-A X-ray crystal structure of an oxidized derivative of the double mutant (S22 1C/P225A) supports the protein design strategy in showing that the P225A mutation partly relieves the steric crowding expected from the S221 C substitution, thus accounting for its improved catalytic efficiency. Stable and synthetically reasonable alkyl ester peptide substrates were prepared that rapidly acylate the S22 1C/P225A enzyme, and aminolysis of the resulting thioacyl-enzyme intermediate by various peptides is strongly preferred over hydrolysis. The efficiency of aminolysis is relatively insensitive to the sequence of the first two residues in the acyl acceptor peptide whose a-amino group attacks the thioacyl-enzyme.To obtain greater flexibility in the choice of coupling sites, a set of three additional peptide ligases were engineered by introducing mutations into the parent ligase (S22 1 C/P225A) that were previously shown to change the specificity of subtilisin for the residue nearest the acyl bond (the P, residue). The specificity properties of the parent ligase and derivatives of it paralleled those of wild type and corresponding specificity variants. The set of specific peptide ligases should be useful for blockwise synthesis or semisynthesis of proteins in aqueous solution.C h e m i c a l approaches for synthesis and engineering of proteins offer many advantages to recombinant methods in that one can incorporate nonnatural or selectively labeled amino acids. However, peptide synthesis is practically limited to small proteins (typically <50 residues) due to the accumulation of side products and racemization that complicate product purification and decrease yields [for recent reviews see Kaiser (1989) and Offord (1987)l.Proteolytic enzymes, in particular serine proteases, have been used as alternatives to synthetic peptide chemistry because of their stereoselective properties and mild reaction conditions [for reviews see Kullmann (1987) and Chaiken (1981)l. Such enzymes have also been used to complement chemical coupling methods and allow larger proteins to be synthesized by blockwise enzymatic coupling of synthetic fragments [Tnouye et al., 1979; for review see Chaiken (1981)l. However, the narrow substrate specificities and hydrolytic activities of serine proteases have limited their use in peptide synthesis.A central problem in the use of serine proteases for peptide synthesis is that hydrolysis of the acyl-enzyme intermediate is strongly favored over aminoly...

A genetic approach is described to clarify the IgG-binding properties of the N-terminal portion of staphylococcal protein A (region E). Several gene fragments, encoding region E or B or protein A, have been cloned and expressed in Escherichia coli. The gene products were purified by IgG-affinity chromatography and subjected to structural and functional analyses. Both fragments can be efficiently purified using this method, suggesting that region B as well as region E has Fc-binding activity. In addition, gene fusions were assembled giving fragments EB and EE, which both showed a divalent Fc-binding. These results demonstrate that protein A consists of five IgG-binding domains. The implications of these findings for the structure of protein-Aimmunoglobulin-G complexes are discussed.Staphylococcal protein A (SpA) plays an important role in molecular biology owing to its specific interaction with the Fc portion of immunoglobulins from many mammals [l -31. Biological responses to SpA include activation of the complement system, hypersensitivity reactions, cell-mediated cytotoxicity, interferon production, activation of polyclonal antibody synthesis and mitogenic stimulation of lymphocytes [4 -81. Many immunological methods have been developed and refined using SpA as a reagent, including immunoprecipitation techniques and double sandwich immunoassays [9, 101. In addition, solid-phase protein A has been used therapeutically to decrease the amount of circulating immunocomplexes in sera [l 13. Treatments involving continuous-flow extracorporal systems have been tried on patients with advanced carcinomas and acquired immunodeficient syndrome (AIDS).Because of its importance as an immunological tool, extensive structural and biochemical studies of the protein A molecule have been performed during the last two decades [12 -181. Fragmentation of the SpA molecule by trypsin digestion followed by purification and amino acids sequence analysis of the fragments suggested a tetrameric structure of the IgG-binding part [14, 151. A model was therefore proposed where SpA consists of four highly homologous Fc-binding domains (D, A, B and C) followed by a C-terminal region X, which anchors the protein to the cell wall of Staphylococcus aureus [14]. However, binding studies suggested that one molecule of intact SpA can only bind two molecules of IgG [I 9) leading to the hypothesis that SpA is functionally divalent in spite of its tetrameric structure [20].Recently a model for the formation of protein A-IgG soluble complexes has been proposed based on the evidence that IgG has two functional binding sites for SpA and that SPA has four functionally binding sites for IgG [21] nucleotide sequence analysis of the cloned SpA gene [22] reveals, however, a fifth region E, homologous to the four repetitive regions earlier identified by protein analysis. Sequence comparison between the repetitive IgG-binding regions show that region E has diverged more than the other four regions (D, A, B and C ) to a hypothetical consensus sequence [23]. ...

11 beta-Hydroxysteroid dehydrogenase type 1 (11 beta-HSD1) has been proposed as a new target for type 2 diabetes drugs. The aim of the present study was to assess the effects of inhibition of 11 beta-HSD1 on blood glucose levels, glucose tolerance, and insulin sensitivity in mouse models of type 2 diabetes. BVT.2733 is an isoform-selective inhibitor of mouse 11 beta-HSD1. Hyperglycemic and hyperinsulinemic ob/ob, db/db, KKAy, and normal C57BL/6J mice were orally administered BVT.2733 (200 mg/kg.d, twice daily). In hyperglycemic, but not in normal mice, BVT.2733 lowered circulating glucose (to 50-88% of control) and insulin (52-65%) levels. In oral glucose tolerance tests in ob/ob and KKAy mice, glucose concentrations were 65-75% of vehicle values after BVT.2733 treatment, and in KKAy mice insulin concentrations were decreased (62-74%). Euglycemic, hyperinsulinemic clamps demonstrated decreased endogenous glucose production (21-61%). Analysis of hepatic mRNA in KKAy mice showed reduced phosphoenolpyruvate carboxykinase mRNA (71%). A slight reduction in food intake was observed in ob/ob and KKAy mice. Cholesterol, triglycerides, and free fatty acid levels were decreased to 81-86% in KKAy mice after a 4-h fast. The results support previous suggestions that selective 11 beta-HSD1 inhibitors lower blood glucose levels and improve insulin sensitivity in different mouse models of type 2 diabetes.

We describe the development of a novel serum albumin binding protein showing an extremely high affinity (K(D)) for HSA in the femtomolar range. Using a naturally occurring 46-residue three-helix bundle albumin binding domain (ABD) of nanomolar affinity for HSA as template, 15 residues were targeted for a combinatorial protein engineering strategy to identify variants showing improved HSA affinities. Sequencing of 55 unique phage display-selected clones showed a strong bias for wild-type residues at nine positions, whereas various changes were observed at other positions, including charge shifts. Additionally, a few non-designed substitutions appeared. On the basis of the sequences of 12 variants showing high overall binding affinities and slow dissociation rate kinetics, a set of seven 'second generation' variants were constructed. One variant denoted ABD035 displaying wild-type-like secondary structure content and excellent thermal denaturation/renaturation properties showed an apparent affinity for HSA in the range of 50-500 fM, corresponding to several orders of magnitude improvement compared with the wild-type domain. The ABD035 variant also showed an improved affinity toward serum albumin from a number of other species, and a capture experiment involving human serum indicated that the selectivity for serum albumin had not been compromised from the affinity engineering.

The human epidermal growth factor receptor 2 (HER2) is specifically overexpressed in tumors of several cancers, including an aggressive form of breast cancer. It is therefore a target for both cancer diagnostics and therapy. The 58 amino acid residue ZHER2 affibody molecule was previously engineered as a high-affinity binder of HER2. Here we determined the structure of ZHER2 in solution and the crystal structure of ZHER2 in complex with the HER2 extracellular domain. ZHER2 binds to a conformational epitope on HER2 that is distant from those recognized by the therapeutic antibodies trastuzumab and pertuzumab. Its small size and lack of interference may provide ZHER2 with advantages for diagnostic use or even for delivery of therapeutic agents to HER2-expressing tumors when trastuzumab or pertuzumab are already employed. Biophysical characterization shows that ZHER2 is thermodynamically stable in the folded state yet undergoing conformational interconversion on a submillisecond time scale. The data suggest that it is the HER2-binding conformation that is formed transiently prior to binding. Still, binding is very strong with a dissociation constant K D ¼ 22 pM, and perfect conformational homogeneity is therefore not necessarily required in engineered binding proteins. A comparison of the original Z domain scaffold to free and bound ZHER2 structures reveals how high-affinity binding has evolved during selection and affinity maturation and suggests how a compromise between binding surface optimization and stability and dynamics of the unbound state has been reached.protein engineering | molecular recognition | protein-protein interactions | protein conformational dynamics | cancer therapy

The Affibody molecule Z HER2:342-pep2 , site-specifically and homogeneously conjugated with a 1,4,7,10-tetra-azacylododecane-N, N ¶,N 00 ,N Ø-tetraacetic acid (DOTA) chelator, was produced in a single chemical process by peptide synthesis. DOTA-Z HER2:342-pep2 folds spontaneously and binds HER2 with 65 pmol/L affinity. Efficient radiolabeling with >95% incorporation of 111 In was achieved within 30 min at low (room temperature) and high temperatures (up to 90°C). Tumor uptake of 111 In-DOTA-Z HER2:342-pep2 was specific for HER2-positive xenografts. A high tumor uptake of 23% injected activity per gram tissue, a tumor-to-blood ratio of >7.5, and high-contrast gamma camera images were obtained already 1 h after injection. Pretreatment with Herceptin did not interfere with tumor targeting, whereas degradation of HER2 using the heat shock protein 90 inhibitor 17-allylaminogeldanamycin before administration of 111In-DOTA-Z HER2:342-pep2 obliterated the tumor image. The present results show that radiolabeled synthetic DOTA-Z HER2:342-pep2 has the potential to become a clinically useful radiopharmaceutical for in vivo molecular imaging of HER2-expressing carcinomas.