Bispecific Antibody Detection Using Antigen-Conjugated Synthetic Nucleic Acid Strands.

We report here the development of two different sensing strategies based on the use of antigen-conjugated nucleic acid strands for the detection of a bispecific antibody against the tumor-related proteins Mucin1 and epidermal growth factor receptor. Both approaches work well in serum samples (nanomolar sensitivity), show high specificity against the two monospecific antibodies, and are rapid. The results presented here demonstrate the versatility of DNA-based platforms for the detection of bispecific antibodies and could represent a versatile alternative to other more reagent-intensive and time-consuming analytical approaches.

Crystal structure of a class III adenylyl cyclase-like ATP-binding protein from Pseudomonas aeruginosa.

In many organisms, the ubiquitous second messenger cAMP is formed by at least one member of the adenylyl cyclase (AC) Class III. These ACs feature a conserved dimeric catalytic core architecture, either through homodimerization or through pseudo-heterodimerization of a tandem of two homologous catalytic domains, C1 and C2, on a single protein chain. The symmetric core features two active sites, but in the C1-C2 tandem one site degenerated into a regulatory center. Analyzing bacterial AC sequences, we identified a Pseudomonas aeruginosa AC-like protein (PaAClp) that shows a surprising swap of the catalytic domains, resulting in an unusual C2-C1 arrangement. We cloned and recombinantly produced PaAClp. The protein bound nucleotides but showed no AC or guanylyl cyclase activity, even in presence of a variety of stimulating ligands of other ACs. Solving the crystal structure of PaAClp revealed an overall structure resembling active class III ACs but pronounced shifts of essential catalytic residues and structural elements. The structure contains a tightly bound ATP, but in a binding mode not suitable for cAMP formation or ATP hydrolysis, suggesting that PaAClp acts as an ATP-binding protein.

KEAP1-modifying small molecule reveals muted NRF2 signaling responses in neural stem cells from Huntington's disease patients.

The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntington's disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology.

A conserved NAD+ binding pocket that regulates protein-protein interactions during aging.

DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD+ (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD+ to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD+ concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD+ Thus, NAD+ directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging.

Development of 1,2,4-Oxadiazoles as Potent and Selective Inhibitors of the Human Deacetylase Sirtuin 2: Structure-Activity Relationship, X-ray Crystal Structure, and Anticancer Activity.

Sirt2 is a target for the treatment of neurological, metabolic, and age-related diseases including cancer. Here we report a series of Sirt2 inhibitors based on the 1,2,4-oxadiazole scaffold. These compounds are potent Sirt2 inhibitors active at single-digit µM level by using the Sirt2 substrate α-tubulin-acetylLys40 peptide and inactive up to 100 µM against Sirt1, -3, and -5 (deacetylase and desuccinylase activities). Their mechanism of inhibition is uncompetitive toward both the peptide substrate and NAD+, and the crystal structure of a 1,2,4-oxadiazole analog in complex with Sirt2 and ADP-ribose reveals its orientation in a still unexplored subcavity useful for further inhibitor development. Tested in leukemia cell lines, 35 and 39 induced apoptosis and/or showed antiproliferative effects at 10 or 25 µM after 48 h. Western blot analyses confirmed the involvement of Sirt2 inhibition for their effects in NB4 and in U937 cells. Our results provide novel Sirt2 inhibitors with a compact scaffold and structural insights for further inhibitor improvement.

1,4-Dihydropyridines Active on the SIRT1/AMPK Pathway Ameliorate Skin Repair and Mitochondrial Function and Exhibit Inhibition of Proliferation in Cancer Cells.

Modulators of sirtuins are considered promising therapeutic targets for the treatment of cancer, cardiovascular, metabolic, inflammatory, and neurodegenerative diseases. Here we prepared new 1,4-dihydropyridines (DHPs) bearing changes at the C2/C6, C3/C5, C4, or N1 position. Tested with the SIRTainty procedure, some of them displayed increased SIRT1 activation with respect to the prototype 3a, high NO release in HaCat cells, and ameliorated skin repair in a mouse model of wound healing. In C2C12 myoblasts, two of them improved mitochondrial density and functions. All the effects were reverted by coadministration of compound C (9), an AMPK inhibitor, or of EX-527 (10), a SIRT1 inhibitor, highlighting the involvement of the SIRT1/AMPK pathway in the action of DHPs. Finally, tested in a panel of cancer cells, the water-soluble form of 3a, compound 8, displayed antiproliferative effects in the range of 8-35 µM and increased H4K16 deacetylation, suggesting a possible role for SIRT1 activators in cancer therapy.

The discovery of a highly selective 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one SIRT2 inhibitor that is neuroprotective in an in vitro Parkinson's disease model.

Sirtuins, NAD(+) -dependent histone deacetylases (HDACs), have recently emerged as potential therapeutic targets for the treatment of a variety of diseases. The discovery of potent and isoform-selective inhibitors of this enzyme family should provide chemical tools to help determine the roles of these targets and validate their therapeutic value. Herein, we report the discovery of a novel class of highly selective SIRT2 inhibitors, identified by pharmacophore screening. We report the identification and validation of 3-((2-methoxynaphthalen-1-yl)methyl)-7-((pyridin-3-ylmethyl)amino)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (ICL-SIRT078), a substrate-competitive SIRT2 inhibitor with a Ki value of 0.62 ± 0.15 µM and more than 50-fold selectivity against SIRT1, 3 and 5. Treatment of MCF-7 breast cancer cells with ICL-SIRT078 results in hyperacetylation of α-tubulin, an established SIRT2 biomarker, at doses comparable with the biochemical IC50 data, while suppressing MCF-7 proliferation at higher concentrations. In concordance with the recent reports that suggest SIRT2 inhibition is a potential strategy for the treatment of Parkinson's disease, we find that compound ICL-SIRT078 has a significant neuroprotective effect in a lactacystin-induced model of Parkinsonian neuronal cell death in the N27 cell line. These results encourage further investigation into the effects of ICL-SIRT078, or an optimised derivative thereof, as a candidate neuroprotective agent in in vivo models of Parkinson's disease.

Molecular architecture of the human protein deacetylase Sirt1 and its regulation by AROS and resveratrol.

Sirtuins are NAD+-dependent protein deacetylases regulating metabolism, stress responses and ageing processes. Among the seven mammalian Sirtuins, Sirt1 is the physiologically best-studied isoform. It regulates nuclear functions such as chromatin remodelling and gene transcription, and it appears to mediate beneficial effects of a low calorie diet which can partly be mimicked by the Sirt1 activating polyphenol resveratrol. The molecular details of Sirt1 domain architecture and regulation, however, are little understood. It has a unique N-terminal domain and CTD (C-terminal domain) flanking a conserved Sirtuin catalytic core and these extensions are assumed to mediate Sirt1-specific features such as homo-oligomerization and activation by resveratrol. To analyse the architecture of human Sirt1 and functions of its N- and C-terminal extensions, we recombinantly produced Sirt1 and Sirt1 deletion constructs as well as the AROS (active regulator of Sirt1) protein. We then studied Sirt1 features such as molecular size, secondary structure and stimulation by small molecules and AROS. We find that Sirt1 is monomeric and has extended conformations in its flanking domains, likely disordered especially in the N-terminus, resulting in an increased hydrodynamic radius. Nevertheless, both termini increase Sirt1 deacetylase activity, indicating a regulatory function. We also find an unusual but defined conformation for AROS protein, which fails, however, to stimulate Sirt1. Resveratrol, in contrast, activates the Sirt1 catalytic core independent of the terminal domains, indicating a binding site within the catalytic core and suggesting that small molecule activators for other isoforms might also exist.

Crystal structure analysis of human Sirt2 and its ADP-ribose complex.

Sirtuins are NAD(+)-dependent protein deacetylases that regulate metabolism and aging-related processes. Sirt2 is the only cytoplasmic isoform among the seven mamalian Sirtuins (Sirt1-7) and structural information concerning this isoform is limited. We crystallized Sirt2 in complex with a product analog, ADP-ribose, and solved this first crystal structure of a Sirt2 ligand complex at 2.3Å resolution. Additionally, we re-refined the structure of the Sirt2 apoform and analyzed the conformational changes associated with ligand binding to derive insights into the dynamics of the enzyme. Our analyses also provide information on Sirt2 peptide substrate binding and structural states of a Sirt2-specific protein region, and our insights and the novel Sirt2 crystal form provide helpful tools for the development of Sirt2 specific inhibitors.

Crystal structures of receptors involved in small molecule transport across membranes.

This paper briefly reviews contemporary protein crystallography and focuses on six receptor proteins of membrane-intrinsic ATP binding cassette (ABC) transporters. Three of these receptors are specific for carbohydrates and three for amino acids. The receptor GacH of the transporter GacFGH from Streptomyces glaucescens is specific for acarbose and its homologs, and MalE of Salmonella typhimurium is specific for maltose but also forms a complex with acarbose, and the third receptor is the highly specific d-galactose receptor AcbH of the transporter AcbFGH from Actinoplanes sp. Concerning the receptors for amino acids, ArtJ belongs to the ArtJ-(MP)(2) transporter of Geobacillus stearotermophilus and recognizes and binds to positively charged arginine, lysine, and histidine with different sizes of side chains, contrasting the receptors Ngo0372 and Ngo2014 from Neisseria gonorrhaeae that are highly specific for cystine and cysteine, respectively. The differences in the rather unspecific receptors GacH, MalE and ArtJ are compared with the highly specific receptors AcbH, Ngo0372 and Ngo2014.

Crystal structures of two solute receptors for L-cystine and L-cysteine, respectively, of the human pathogen Neisseria gonorrhoeae.

ATP-binding cassette (ABC) transporters are integral membrane proteins that carry a variety of substrates across biological membranes at the expense of ATP. The here considered prokaryotic canonical importers consist of three entities: an extracellular solute receptor, two membrane-intrinsic proteins forming a translocation pathway, and two cytoplasmic ATP-binding subunits. The ngo0372-74 and ngo2011-14 gene clusters from the human pathogen Neisseria gonorrhoeae were predicted by sequence homology as ABC transporters for the uptake of cystine and cysteine, respectively, and chosen for structural characterization. The structure of the receptor component Ngo0372 was obtained in a ligand-free "open" conformation and in a "closed" conformation when co-crystallized with L-cystine. Our data provide the first structural information of an L-cystine ABC transporter. Dissociation constants of 21 and 33 nM for L-cystine and L-selenocystine, respectively, were determined by isothermal titration calorimetry. In contrast, L-cystathionine and L-djenkolic acid are weak binders, while no binding was detectable for S-methyl-L-cysteine. Mutational analysis of two residues from the binding pocket, Trp97 and Tyr59, revealed that the latter is crucial for L-cystine binding. The structure of the Ngo2014 receptor was obtained in closed conformation in complex with co-purified L-cysteine. The protein binds L-cysteine with a K(d) of 26 nM. Comparison of the structures of both receptors and analysis of the ligand binding sites shed light on the mode of ligand recognition and provides insight into the tight binding of both substrates. Moreover, since L-cystine limitation leads to reduction in virulence of N. gonorrhoeae, Ngo0372 might be suited as target for an antimicrobial vaccine.