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Background:The EGF receptor (EGFR) is an important therapeutic target. Results: Bispecific anti-EGFR designed ankyrin repeat proteins (DARPins), alternative targeting molecules efficiently produced in bacteria, were shown to inhibit A431 cell proliferation and receptor recycling. Conclusion: One bispecific construct containing four DARPins showed a biological activity superior to that of the registered antibody cetuximab. Significance: Bispecific DARPins may form building blocks for tomorrow's cancer therapeutics.

The enzyme vascular non-inflammatory molecule-1 (vanin 1) is highly expressed at gene and protein level in many organs, such as the liver, intestine, and kidney. Its major function is related to its pantetheinase activity; vanin 1 breaks down pantetheine in cysteamine and pantothenic acid, a precursor of coenzyme A. Indeed, its physiological role seems strictly related to coenzyme A metabolism, lipid metabolism, and energy production. In recent years, many studies have elucidated the role of vanin 1 under physiological conditions in relation to oxidative stress and inflammation. Vanin’s enzymatic activity was found to be of key importance in certain diseases, either for its protective effect or as a sensitizer, depending on the diseased organ. In this review, we discuss the role of vanin 1 in the liver, kidney, intestine, and lung under physiological as well as pathophysiological conditions. Thus, we provide a more complete understanding and overview of its complex function and contribution to some specific pathologies.

An optimized HPLC method with photodiode array detection was developed and applied to analyse the curcuminoids curcumin, demethoxycurcumin, and bis-demethoxycurcumin in rhizomes of Curcuma mangga Val &. v. Zijp, C. heyneana Val. & v. Zijp, C. aeruginosa Roxb. and C. soloensis Val. (Zingiberaceae), indigenous to Indonesia. The method was validated with an isocratic system, a short run time of 10 min and a baseline separation. The curcuminoid content was 0.18-0.47% for C. mangga, 0.98-3.21% for C. heyneana, 0.02-0.03% for C. aeruginosa and 0.40% for C. soloensis.

Enzymes have become an attractive alternative to conventional catalysts in numerous industrial processes. However, their properties do not always meet the criteria of the application of interest. Directed evolution is a powerful tool for adapting the characteristics of an enzyme. However, selection of the evolved variants is a critical step, and therefore new strategies to enable selection of the desired enzymatic activity have been developed. This review focuses on these novel strategies for selecting enzymes from large libraries, in particular those that are used in the synthesis of pharmaceutical intermediates and pharmaceuticals.

Phage display can be used as a protein‐engineering tool for the selection of proteins with desirable binding properties from a library of mutants. Here we describe the application of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has important properties for the preparation of the pharmaceutically relevant chiral compound 1,2‐O‐isopropylidene‐sn‐glycerol (IPG). PCR mutagenesis with spiked oligonucleotides was employed for saturation mutagenesis of a stretch of amino acids near the active site. After expression of these mutants on bacteriophages, dual selection with (S)‐(+)‐ and (R)‐(−)‐IPG stereoisomers covalently coupled to enantiomeric phosphonate suicide inhibitors (SIRAN Sc and Rc inhibitors, respectively) was used for the isolation of variants with inverted enantioselectivity. The mutants were further characterised by determination of their Michaelis–Menten parameters. The 3D structures of the Sc and Rc inhibitor–lipase complexes were determined and provided structural insight into the mechanism of enantioselectivity of the enzyme. In conclusion, we have used phage display as a fast and reproducible method for the selection of Bacillus lipase A mutant enzymes with inverted enantioselectivity.

Although part of the coenzyme A pathway, vanin 1 (also known as pantetheinase) sits on the cell surface of many cell types as an ectoenzyme, catalyzing the breakdown of pantetheine to pantothenic acid (vitamin B5) and cysteamine, a strong reducing agent. Vanin 1 was initially discovered as a protein involved in the homing of leukocytes to the thymus. Numerous studies have shown that vanin 1 is involved in inflammation, and more recent studies have shown a key role in metabolic disease. Here, the X-ray crystal structure of human vanin 1 at 2.25 Å resolution is presented, which is the first reported structure from the vanin family, as well as a crystal structure of vanin 1 bound to a specific inhibitor. These structures illuminate how vanin 1 can mediate its biological roles by way of both enzymatic activity and protein-protein interactions. Furthermore, it sheds light on how the enzymatic activity is regulated by a novel allosteric mechanism at a domain interface.

The epidermal growth factor receptor (EGFR) is a member of the ErbB family that can promote the migration and proliferation of breast cancer cells. Therapies that target EGFR can promote the dimerization of EGFR with other ErbB receptors, which is associated with the development of drug resistance. Understanding how interactions among ErbB receptors alter EGFR biology could provide avenues for improving cancer therapy. We found that EGFR interacted directly with the CYT1 and CYT2 variants of ErbB4 and the membrane-anchored intracellular domain (mICD). The CYT2 variant, but not the CYT1 variant, protected EGFR from ligand-induced degradation by competing with EGFR for binding to a complex containing the E3 ubiquitin ligase c-Cbl and the adaptor Grb2. Cultured breast cancer cells overexpressing both EGFR and ErbB4 CYT2 mICD exhibited increased migration. With molecular modeling, we identified residues involved in stabilizing the EGFR dimer. Mutation of these residues in the dimer interface destabilized the complex in cells and abrogated growth factor-stimulated cell migration. An exon array analysis of 155 breast tumors revealed that the relative mRNA abundance of the ErbB4 CYT2 variant was increased in ER+ HER2- breast cancer patients, suggesting that our findings could be clinically relevant. We propose a mechanism whereby competition for binding to c-Cbl in an ErbB signaling heterodimer promotes migration in response to a growth factor gradient.