Small Changes Make the Difference for SIRT2: Two Different Binding Modes for 3-Arylmercapto-Acylated Lysine Derivatives.

Sirtuins are protein deacylases regulating metabolism and stress responses and implicated in aging-related diseases. Modulators of the human sirtuins 1-7 are sought as chemical tools and potential therapeutics, for example, for treatment of cancer. We were able to show that 3-aryl-mercapto-succinylated- and 3-benzyl-mercapto-succinylated peptide derivatives yield selective Sirt5 inhibitors with low nM Ki values. Here, we synthesized and characterized 3-aryl-mercapto-butyrylated peptide derivatives as effective and selective sirtuin 2 inhibitors with KD values in the low nanomolar range. According to kinetic measurements and microscale thermophoresis/surface plasmon resonance experiments, the respective inhibitors bind with the 3-aryl-mercapto moiety in the selectivity pocket of Sirtuin 2, inducing a rearrangement of the active site. In contrast, 3-aryl-mercapto-nonalyl or palmitoyl derivatives are characterized by a switch in the binding mode blocking both the hydrophobic channel by the fatty acyl chain and the nicotinamide pocket by the 3-aryl-mercapto moiety.

Synthesis of Multiple Bispecific Antibody Formats with Only One Single Enzyme Based on Enhanced Trypsiligase.

Bispecific antibodies (bsAbs) were first developed in the 1960s and are now emerging as a leading class of immunotherapies for cancer treatment with the potential to further improve clinical efficacy and safety. Many different formats of bsAbs have been established in the last few years, mainly generated genetically. Here we report on a novel, flexible, and fast chemo-enzymatic, as well as purely enzymatic strategies, for generating bispecific antibody fragments by covalent fusion of two functional antibody Fab fragments (Fabs). For the chemo-enzymatic approach, we first modified the single Fabs site-specifically with click anchors using an enhanced Trypsiligase variant (eTl) and afterward converted the modified Fabs into the final heterodimers via click chemistry. Regarding the latter, we used the strain-promoted alkyne-azide cycloaddition (SPAAC) and inverse electron-demand Diels-Alder reaction (IEDDA) click approaches well known for their fast reaction kinetics and fewer side reactions. For applications where the non-natural linkages or hydrophobic click chemistry products might interfere, we developed two purely enzymatic alternatives enabling C- to C- and C- to N-terminal coupling of the two Fabs via a native peptide bond. This simple system could be expanded into a modular system, eliminating the need for extensive genetic engineering. The bispecific Fab fragments (bsFabs) produced here to bind the growth factors ErbB2 and ErbB3 with similar KD values, such as the sole Fabs. Tested in breast cancer cell lines, we obtained biologically active bsFabs with improved properties compared to its single Fab counterparts.

Trypsiligase-Catalyzed Labeling of Proteins on Living Cells.

Fluorescent fusion proteins are powerful tools for studying biological processes in living cells, but universal application is limited due to the voluminous size of those tags, which might have an impact on the folding, localization or even the biological function of the target protein. The designed biocatalyst trypsiligase enables site-directed linkage of small-sized fluorescence dyes on the N terminus of integral target proteins located in the outer membrane of living cells through a stable native peptide bond. The function of the approach was tested by using the examples of covalent derivatization of the transmembrane proteins CD147 as well as the EGF receptor, both presented on human HeLa cells. Specific trypsiligase recognition of the site of linkage was mediated by the dipeptide sequence Arg-His added to the proteins' native N termini, pointing outside the cell membrane. The labeling procedure takes only about 5 minutes, as demonstrated for couplings of the fluorescence dye tetramethyl rhodamine and the affinity label biotin as well.

Backbone and nearly complete side-chain chemical shift assignments of the human death-associated protein 1 (DAP1).

Death-associated protein 1 (DAP1) is a proline-rich cytoplasmatic protein highly conserved in most eukaryotes. It has been reported to be involved in controlling cell growth and migration, autophagy and apoptosis. The presence of human DAP1 is associated to a favourable prognosis in different types of cancer. Here we describe the almost complete [Formula: see text], [Formula: see text], and [Formula: see text] chemical shift assignments of the human DAP1. The limited spectral dispersion, mainly in the [Formula: see text] region, and the lack of defined secondary structure elements, predicted based on chemical shifts, identifies human DAP1 as an intrinsically disordered protein (IDP). This work lays the foundation for further structural investigations, dynamic studies, mapping of potential interaction partners or drug screening and development.

1H, 13C, and 15N Backbone assignments of the human brain and acute leukemia cytoplasmic (BAALC) protein.

The brain and acute leukemia cytoplasmic (BAALC; UniProt entry Q8WXS3) is a 180-residue-long human protein having six known isoforms. BAALC is expressed in either hematopoietic or neuroectodermal cells and its specific function is still to be revealed. However, as a presumably membrane-anchored protein at the cytoplasmic side it is speculated that BAALC exerts its function at the postsynaptic densities of certain neurons and might play a role in developing cytogenetically normal acute myeloid leukemia (CN-AML) when it is highly overexpressed by myeloid or lymphoid progenitor cells. In order to better understand the physiological role of BAALC and to provide the basis for a further molecular characterization of BAALC, we report here the 1H, 13C, and 15N resonance assignments for the backbone nuclei of its longest hematopoietic isoform (isoform 1). In addition, we present a 1HN and 15NH chemical shift comparison of BAALC with its shortest, neuroectodermal isoform (isoform 6) which shows only minor changes in the 1H and 15N chemical shifts.

Site-Specific Modification of Proteins via Trypsiligase.

Site-specific incorporation of artificial functionalities into protein targets is an important tool in both basic and applied research and can be a major challenge to protein chemists. Chemical labeling methods often targeting multiple positions within a protein and therefore suffer from lack of specificity. Enzymatic protein modification is an attractive alternative due to the inherent regioselectivity and stereoselectivity of enzymes. In this contribution we describe the application of the highly specific trypsin variant named trypsiligase for the site-specific modification of virtual any target protein. We present two general routes of modification resulting in either N- or C-terminal functionalized protein products. Both reaction regimes proceed under mild and bioorthogonal conditions in a short period of time which result in homogeneously modified proteins bearing the artificial functionality exclusively at the desired position. We detail protocols for the expression and purification of trypsiligase as well as the construction of peptide or acyl donor ester probes used as substrates for the biocatalyst. In addition, we provide instructions how to perform the ultimate bioconjugation reactions and finally render assistance for the qualitative and quantitative analysis of the reaction course and outcome.

Trypsiligase-Catalyzed Peptide and Protein Ligation.

Site-specific incorporation of nonproteinogenic functionalities into protein targets is an important tool in both basic and applied research and represents a major challenge to protein chemists. Chemical labeling methods often target multiple positions within a protein and therefore suffer from a lack of specificity. Enzymatic protein modification is an attractive alternative due to the inherent regioselectivity and stereoselectivity of enzymes. In this chapter we describe the application of the highly specific trypsin variant trypsiligase for the site-specific modification of virtual any target protein. We present two general routes of modification resulting in either N- or C-terminal functionalized protein products. Reactions rapidly proceed under mild conditions and result in homogeneously modified proteins bearing the artificial functionality exclusively at the desired position. We detail protocols for the expression and purification of trypsiligase as well as the synthesis of peptide (ester) substrates. In addition, we provide instructions for the bioconjugation reactions and for the qualitative and quantitative analysis of reaction progress and efficiency.

Potent and Selective Inhibitors of Human Sirtuin 5.

Sirtuins are protein deacylases that regulate metabolism and stress responses and are implicated in aging-related diseases. Modulators of the human sirtuins Sirt1-7 are sought as chemical tools and potential therapeutics, e.g., for cancer. Selective and potent inhibitors are available for Sirt2, but selective inhibitors for Sirt5 with Ki values in the low nanomolar range are lacking. We synthesized and screened 3-arylthiosuccinylated and 3-benzylthiosuccinylated peptide derivatives yielding Sirt5 inhibitors with low-nanomolar Ki values. A biotinylated derivative with this scaffold represents an affinity probe for human Sirt5 that is able to selectively extract this enzyme out of complex biological samples like cell lysates. Crystal structures of Sirt5/inhibitor complexes reveal that the compounds bind in an unexpected manner to the active site of Sirt5.

Selective Coupling of Click Anchors to Proteins via Trypsiligase.

The combination of pure chemical methods with enzymatic approaches offers a kit system with maximum flexibility for site-specifically tagging proteins with a broad variety of artificial structures. Trypsiligase, a recently introduced designer enzyme for both N- and C-terminal site-specific labeling of peptides and proteins, has been used to introduce click anchors into the human protein cyclophilin 18 and the antibody Fab fragments anti-TNFα and anti-Her2. The subsequent click reactions with tetrazine or norbornene moieties lead to quantitative conversions to the corresponding dihydropyridazine products, thereby forming a stable covalent linkage between the label and the protein of interest. With this technology, cyclophilin 18 has been efficiently modified with the fluorescent dansyl moiety and the pharmaceutically relevant polymer PEG exclusively at its N-terminus. With the same methodology, the Fab fragments of anti-TNFα and anti-Her2 were derivatized exclusively at their C-terminal ends with PEG and the fluorescent dye carboxyfluorescein in the case of anti-TNFα or with the cytotoxic payload DM1 in the case of anti-Her2, to form a homogeneous antibody-drug conjugate (ADC).

Derivatization of antibody Fab fragments: a designer enzyme for native protein modification.

Bioconjugates, such as antibody-drug conjugates, have gained recent attention because of their increasing use in therapeutic and diagnostic applications. Commonly used conjugation reactions based upon chemoselective reagents exhibit a number of drawbacks: most of these reactions lack regio- and stereospecificity, thus resulting in loss of protein functionality due to random modifications. Enzymes provide an obvious solution to this problem, but the intrinsic (natural) substrate specificities of existing enzymes pose severe limitations to the kind of modifications that can be introduced. Here we describe the application of the novel trypsin variant trypsiligase for site-specific modification of the C terminus of a Fab antibody fragment via a stable peptide bond. The suitability of this designed biocatalyst was demonstrated by coupling the Her2-specific Fab to artificial functionalities of either therapeutic (PEG) or diagnostic (fluorescein) relevance. In both cases we obtained homogeneously modified Fab products bearing the artificial functionality exclusively at the desired position.

On the nature of interactions between ionic liquids and small amino-acid-based biomolecules.

During the last decade, ionic liquids (ILs) have revealed promising properties and applications in many research fields, including biotechnology and biological sciences. The focus of this contribution is to give a critical review of the phenomena observed and current knowledge of the interactions occurring on a molecular basis. As opposed to the huge advances made in understanding the properties of proteins in ILs, complementary investigations dealing with interactions between ILs and peptides or oligopeptides are underrepresented and are mostly only of phenomenological nature. However, the field has received more attention in the last few years. This Review features a meta-analysis of the available data and findings and should, therefore, provide a basis for a scientifically profound understanding of the nature and mechanisms of interactions between ILs and structured or nonstructured peptides. Fundamental aspects of the interactions between different peptides/oligopeptides and ILs are complemented by sections on the experimental (spectroscopy, structural biology) and theoretical (computational chemistry) possibilities to explain the phenomena reported so far in the literature. In effect, this should lead to the development of novel applications and support the understanding of IL-solute interactions in general.

Reverse proteolysis promoted by in situ generated peptide ester fragments.

In this contribution we describe a general synthesis concept for the in situ preparation of protease specific reactants using methyl thioesters as universal precursors. The precursor esters are readily available by standard synthesis procedures and can be used directly as reactants for protease-mediated peptide coupling reactions. Alternatively, they can serve as initial building blocks for the in situ preparation of various types of substrate mimetics. The synthesis of the latter is achieved by a one-pot spontaneous transthioesterification reaction of the parent thioester (Y-(Xaa)(n)-SMe-->Y-(Xaa)(n)-SR; R: CH(2)CH(2)COOH, CH(2)C(6)H(5), C(6)H(4)NHC(:NH)NH(2)), which proceeds efficiently in both a sequential manner and parallel to the subsequent enzymatic reaction. The resulting substrate mimetics act as efficient acyl donor components and show the typical behavior of substrate mimicry enabling irreversible reactions with originally nonspecific acyl moieties. Neither a workup of the substrate mimetic intermediate nor changes of the reaction conditions during the whole synthesis process are required. Model peptide syntheses using trypsin, alpha-chymotrypsin, and V8 protease as the biocatalysts proved the function of the approach and illustrated its synthetic value for protease-mediated reactions and the compatibility of the approach with state-of-the-art solid-phase peptide ester synthesis methods.

Substrate mimetics-specific peptide ligases: studies on the synthetic utility of a zymogen and zymogen-like enzymes.

Although proteases are capable of synthesizing peptide bonds, they are not proficient at peptide fragment ligation. Further manipulations are needed to shift the native enzyme activity from the cleavage to the synthesis of peptides. This account reports on the synthetic potential of nonactivatable trypsinogen and zymogen-like enzymes designed to minimize proteolytic side reactions during peptide synthesis.