Total Chemical Synthesis of LC3A and LC3B Activity-Based Probes.

Autophagy is a conserved cellular process involved in the degradation of intercellular materials. During this process, double-membrane vesicles called autophagosomes engulf cytoplasmic components ready for degradation. A key component in the formation of autophagosomes are the autophagy-related (Atg) proteins, including microtubule-associated protein light chain 3A (LC3A) and 3B (LC3B). After the C-terminus of LC3 is conjugated to a phospholipid, it promotes the elongation of the phagosome and provides a docking station for the delivery of proteins ready for degradation. Since dysregulation of the autophagy pathway has been associated with a variety of human diseases, components of this process have been considered as potential therapeutic targets. However, the mechanistic details of LC3-specific ligases and deconjugation enzymes are far from unraveled and chemical tools for activity profiling could aid in affording more insights into this process. Herein, we describe a native chemical ligation approach for the synthesis of two LC3 activity-based probes (ABPs). Initial studies show that the probes covalently interact with the cysteine protease ATG4B, showcasing the potential of these probes to unravel mechanistic and structural details.

Total Linear Chemical Synthesis of LC3A and LC3B.

Solid-phase peptide synthesis (SPPS) enables the synthesis of chemically modified peptides and proteins. Chemically synthesized ubiquitin(-like) proteins containing a fluorescent tag or reactive warhead have proven to be important tools in elucidating biological processes. Here, we describe the first fully synthetic method for the linear synthesis of two LC3 ubiquitin-like proteins using disaggregating building blocks and heated synthesis. Both LC3A and LC3B were synthesized and equipped with a fluorescent rhodamine tag, followed by folding of the proteins and liquid chromatography-mass spectrometry and SDS-PAGE analysis to prove that the quality of the synthetic material is comparable to expressed material.

In-Plate Chemical Synthesis of Isopeptide-Linked SUMOylated Peptide Fluorescence Polarization Reagents for High-Throughput Screening of SENP Preferences.

Small ubiquitin-like modifiers (SUMOs) are conjugated to protein substrates in cells to regulate their function. The attachment of SUMO family members SUMO1-3 to substrate proteins is reversed by specific isopeptidases called SENPs (sentrin-specific protease). Whereas SENPs are SUMO-isoform or linkage type specific, comprehensive analysis is missing. Furthermore, the underlying mechanism of SENP linkage specificity remains unclear. We present a high-throughput synthesis of 83 isopeptide-linked SUMO-based fluorescence polarization reagents to study enzyme preferences. The assay reagents were synthesized via a native chemical ligation-desulfurization protocol between 11-mer peptides containing a γ-thiolysine and a SUMO3 thioester. Subsequently, five recombinantly expressed SENPs were screened using these assay reagents to reveal their deconjugation activity and substrate preferences. In general, we observed that SENP1 is the most active and nonselective SENP while SENP6 and SENP7 show the least activity. Furthermore, SENPs differentially process peptides derived from SUMO1-3, who form a minimalistic representation of diSUMO chains. To validate our findings, five distinct isopeptide-linked diSUMO chains were chemically synthesized and proteolysis was monitored using a gel-based read-out.

Total Chemical Synthesis of a Functionalized GFP Nanobody.

Chemical protein synthesis has proven to be a powerful tool to access homogenously modified proteins. The chemical synthesis of nanobodies (Nb) would create possibilities to design tailored Nbs with a range of chemical modifications such as tags, linkers, reporter groups, and subsequently, Nb-drug conjugates. Herein, we describe the total chemical synthesis of a 123 amino-acid Nb against GFP. A native chemical ligation- desulfurization strategy was successfully applied for the synthesis of this GFP Nb, modified with a propargyl (PA) moiety for on-demand functionalization. Biophysical characterization indicated that the synthetic GFP Nb-PA was correctly folded after internal disulfide bond formation. The synthetic Nb-PA was functionalized with a biotin or a sulfo-cyanine5 dye by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), resulting in two distinct probes used for functional in vitro validation in pull-down and confocal microscopy settings.

Tissue Distribution and Receptor Activation by Somapacitan, a Long Acting Growth Hormone Derivative.

Somapacitan is a long-acting, once-weekly, albumin-binding growth hormone (GH) derivative. The reversible albumin-binding properties leads to prolonged circulation half-life. Here, we investigated and compared somapacitan with human GH on downstream receptor signaling in primary hepatocytes and hepatocellular models and using isothermal titration calorimetry to characterize receptor binding of somapacitan in the presence or absence of human serum albumin (HSA). With non-invasive fluorescence imaging we quantitatively visualize and compare the temporal distribution and examine the tissue-specific growth hormone receptor (GHR) activation at distribution sites. We found that signaling kinetics were slightly more rapid and intense for GH compared with somapacitan. Receptor binding isotherms were characterized by a high and a low affinity interaction site with or without HSA. Using in vivo optical imaging we found prolonged systemically biodistribution of somapacitan compared with GH, which correlated with plasma pharmacokinetics. Ex vivo mouse organ analysis revealed that the temporal fluorescent intensity in livers dosed with somapacitan was significantly increased compared with GH-dosed livers and correlated with the degree of downstream GHR activation. Finally, we show that fluorescent-labeled analogs distributed to the hypertrophic zone in the epiphysis of proximal tibia of hypophysectomized rats and that somapacitan and GH activate the GHR signaling in epiphyseal tissues.

Enhanced potency of recombinant factor VIIa with increased affinity to activated platelets.

BACKGROUND: Recombinant factor VIIa (rFVIIa) enhances thrombin generation in a platelet-dependent manner; however, rFVIIa binds activated platelets with relatively low affinity. Triggering receptor expressed on myeloid cells (TREM)-like transcript (TLT)-1 is expressed exclusively on activated platelets. OBJECTIVE: To enhance the potency of rFVIIa via binding TLT-1. METHODS: Recombinant FVIIa was conjugated to a TLT-1 binding Fab. In vitro potency of this platelet-targeted rFVIIa (PT-rFVIIa) was evaluated using factor X activation assays and by measuring viscoelastic changes in whole blood. In vivo potency was evaluated using a tail vein transection model in F8-/- mice expressing human TLT-1. RESULTS: PT-rFVIIa and rFVIIa had similar dissociation constant values for tissue factor binding and similar tissue factor-dependent factor X activation. However, PT-rFVIIa had increased catalytic efficiency on TLT-1-loaded vesicles and activated platelets. The in vitro potency in normal human blood with antibody-induced hemophilia A was dependent on assay conditions used; with maximally activated platelets, the half maximal effective concentration for clot time for PT-rFVIIa was 49-fold lower compared with rFVIIa. In the murine bleeding model, a 53-fold lower half maximal effective concentration was observed for blood loss for PT-rFVIIa, supporting the relevance of the assay conditions with maximally activated platelets. In vitro analysis of blood from subjects with hemophilia A confirmed the data obtained with normal blood. CONCLUSIONS: Increasing the affinity of rFVIIa to activated platelets resulted in approximately 50-fold increased potency both in vitro and in the mouse model. The correlation of in vivo with in vitro data using maximally activated platelets supports that these assay conditions are relevant when evaluating platelet-targeted hemostatic concepts.

Long-Acting Human Growth Hormone Analogue by Noncovalent Albumin Binding.

The present work describes a series of human growth hormone (hGH) albumin binder conjugates with an extended in vivo half-life. A broad range of different conjugates were studied by varying the albumin binder structure and conjugation site. Conjugates were conveniently obtained by reductive alkylation or by alkylation to introduced cysteines using functionalized albumin-binding side chains. In vitro and in vivo profiling provided the basis for identification of position L101C in human growth hormone as the most optimal position for conjugation, where both a sufficient level of receptor binding and a suitably long half-life could yield a molecule with potential for a once-weekly dosing regimen.

Engineering of a membrane-triggered activity switch in coagulation factor VIIa.

Recombinant factor VIIa (FVIIa) variants with increased activity offer the promise to improve the treatment of bleeding episodes in patients with inhibitor-complicated hemophilia. Here, an approach was adopted to enhance the activity of FVIIa by selectively optimizing substrate turnover at the membrane surface. Under physiological conditions, endogenous FVIIa engages its cell-localized cofactor tissue factor (TF), which stimulates activity through membrane-dependent substrate recognition and allosteric effects. To exploit these properties of TF, a covalent complex between FVIIa and the soluble ectodomain of TF (sTF) was engineered by introduction of a nonperturbing cystine bridge (FVIIa Q64C-sTF G109C) in the interface. Upon coexpression, FVIIa Q64C and sTF G109C spontaneously assembled into a covalent complex with functional properties similar to the noncovalent wild-type complex. Additional introduction of a FVIIa-M306D mutation to uncouple the sTF-mediated allosteric stimulation of FVIIa provided a final complex with FVIIa-like activity in solution, while exhibiting a two to three orders-of-magnitude increase in activity relative to FVIIa upon exposure to a procoagulant membrane. In a mouse model of hemophilia A, the complex normalized hemostasis upon vascular injury at a dose of 0.3 nmol/kg compared with 300 nmol/kg for FVIIa.

Chemoenzymatic synthesis and in situ application of S-adenosyl-L-methionine analogs.

Analogs of S-adenosyl-L-methionine (SAM) are increasingly applied to the methyltransferase (MT) catalysed modification of biomolecules including proteins, nucleic acids, and small molecules. However, SAM and its analogs suffer from an inherent instability, and their chemical synthesis is challenged by low yields and difficulties in stereoisomer isolation and inhibition. Here we report the chemoenzymatic synthesis of a series of SAM analogs using wild-type (wt) and point mutants of two recently identified halogenases, SalL and FDAS. Molecular modelling studies are used to guide the rational design of mutants, and the enzymatic conversion of L-Met and other analogs into SAM analogs is demonstrated. We also apply this in situ enzymatic synthesis to the modification of a small peptide substrate by protein arginine methyltransferase 1 (PRMT1). This technique offers an attractive alternative to chemical synthesis and can be applied in situ to overcome stability and activity issues.

Binding inhibitors of the bacterial sliding clamp by design.

The bacterial replisome is a target for the development of new antibiotics to combat drug resistant strains. The ß(2) sliding clamp is an essential component of the replicative machinery, providing a platform for recruitment and function of other replisomal components and ensuring polymerase processivity during DNA replication and repair. A single binding region of the clamp is utilized by its binding partners, which all contain conserved binding motifs. The C-terminal Leu and Phe residues of these motifs are integral to the binding interaction. We acquired three-dimensional structural information on the binding site in ß(2) by a study of the binding of modified peptides. Development of a three-dimensional pharmacophore based on the C-terminal dipeptide of the motif enabled identification of compounds that on further development inhibited α-ß(2) interaction at low micromolar concentrations. We report the crystal structure of the complex containing one of these inhibitors, a biphenyl oxime, bound to ß(2), as a starting point for further inhibitor design.

Transglutaminase-mediated methods for site-selective modification of human growth hormone.

Two methods for the site-selective modification of native human growth hormone (hGH) using microbial transglutaminase were developed. In the first method, 1,3-bisaminoxypropane was attached to hGH, providing a direct incorporation of reactive aminoxy groups for further modification. The reaction was shown to be selective for Gln(141), with minor modification at Gln(40). In the second method, modified glutamine substrates were developed for attachment to Lys(145) in hGH. A series of glutamine-substrates were screened, and it was shown that microbial transglutaminase was selective towards substitutions on the glutamine core structure. Products from both methods could be transformed to site selectively mono-PEGylated hGH-derivatives in good isolated yield.

Inhibition of protein interactions with the beta 2 sliding clamp of Escherichia coli DNA polymerase III by peptides from beta 2-binding proteins.

The sliding clamp of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding clamp, a homodimer of the beta subunit, at a single site on the dimer. The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair.