First Generic Teriparatide: Structural and Biological Sameness to Its Reference Medicinal Product.

Teriparatide is an anabolic peptide drug indicated for the treatment of osteoporosis. Recombinant teriparatide was first approved in 2002 and has since been followed by patent-free alternatives under biosimilar or hybrid regulatory application. The aim of this study is to demonstrate the essential similarity between synthetic teriparatide BGW and the reference medicinal product (RMP), and thus to ensure the development of the first generic teriparatide drug. Hence, an extensive side-by-side comparative exercise, focusing on structural and biological activity, was performed using a wide range of state-of-the-art orthogonal methods. Nuclear magnetic resonance (NMR), ion mobility-mass spectrometry (IM-MS), UV, circular dichroism (CD) and Fourier transform infrared (FTIR) demonstrated the structural similarity between teriparatide BGW and the RMP. Comparative cell-based bioassays showed that the synthetic and recombinant peptides have identical behaviors. Teriparatide BGW, as a generic drug, provides an available treatment option for patients with osteoporosis and offers clinical benefits identical to those provided by the RMP.

How Do Cancer-Related Mutations Affect the Oligomerisation State of the p53 Tetramerisation Domain?

Tumour suppressor p53 plays a key role in the development of cancer and has therefore been widely studied in recent decades. While it is well known that p53 is biologically active as a tetramer, the tetramerisation mechanism is still not completely understood. p53 is mutated in nearly 50% of cancers, and mutations can alter the oligomeric state of the protein, having an impact on the biological function of the protein and on cell fate decisions. Here, we describe the effects of a number of representative cancer-related mutations on tetramerisation domain (TD) oligomerisation defining a peptide length that permits having a folded and structured domain, thus avoiding the effect of the flanking regions and the net charges at the N- and C-terminus. These peptides have been studied under different experimental conditions. We have applied a variety of techniques, including circular dichroism (CD), native mass spectrometry (MS) and high-field solution NMR. Native MS allows us to detect the native state of complexes maintaining the peptide complexes intact in the gas phase; the secondary and quaternary structures were analysed in solution by NMR, and the oligomeric forms were assigned by diffusion NMR experiments. A significant destabilising effect and a variable monomer population were observed for all the mutants studied.

Modulation of Functional Phosphorylation Sites by Basic Residues in the Unique Domain of c-Src.

In contrast to the well-studied canonical regulatory mechanisms, the way by which the recently discovered Src N-terminal regulatory element (SNRE) modulates Src activity is not yet well understood. Phosphorylation of serine and threonine residues modulates the charge distribution along the disordered region of the SNRE and may affect a fuzzy complex with the SH3 domain that is believed to act as an information transduction element. The pre-existing positively charged sites can interact with the newly introduced phosphate groups by modulating their acidity, introducing local conformational restrictions, or by coupling various phosphosites into a functional unit. In this paper, we use pH-dependent NMR measurements combined with single point mutations to identify the interactions of basic residues with physiologically important phosphorylated residues and to characterize the effect of these interactions in neighbor residues, thus providing insight into the electrostatic network in the isolated disordered regions and in the entire SNRE. From a methodological point of view, the linear relationships observed between the mutation-induced pKa changes of the phosphate groups of phosphoserine and phosphothreonine and the pH-induced chemical shifts of the NH groups of these residues provide a very convenient alternative to identify interacting phosphate groups without the need to introduce point mutations on specific basic residues.

Linker-Free Synthesis of Antimicrobial Peptides Using a Novel Cleavage Reagent: Characterisation of the Molecular and Ionic Composition by nanoESI-HR MS.

The efficient preparation of novel bioactive peptide drugs requires the availability of reliable and accessible chemical methodologies together with suitable analytical techniques for the full characterisation of the synthesised compounds. Herein, we describe a novel acidolytic method with application to the synthesis of cyclic and linear peptides involving benzyl-type protection. The process consists of the in situ generation of anhydrous hydrogen bromide and a trialkylsilyl bromide that acts as protic and Lewis acid reagents. This method proved to be useful to effectively remove benzyl-type protecting groups and cleave Fmoc/tBu assembled peptides directly attached to 4-methylbenzhydrylamine (MBHA) resins with no need for using mild trifluoroacetic acid labile linkers. The novel methodology was successful in synthesising three antimicrobial peptides, including the cyclic compound polymyxin B3, dusquetide, and RR4 heptapeptide. Furthermore, electrospray mass spectrometry (ESI-MS) is successfully used for the full characterisation of both the molecular and ionic composition of the synthetic peptides.

Proteomic Tools for the Quantitative Analysis of Artificial Peptide Libraries: Detection and Characterization of Target-Amplified PD-1 Inhibitors.

We report a quantitative proteomics data analysis pipeline, which coupled to protein-directed dynamic combinatorial chemistry (DDC) experiments, enables the rapid discovery and direct characterization of protein-protein interaction (PPI) modulators. A low-affinity PD-1 binder was incubated with a library of >100 D-peptides under thiol-exchange favoring conditions, in the presence of the target protein PD-1, and we determined the S-linked dimeric species that resulted, amplified in the protein samples versus the controls. We chemically synthesized the target dimer candidates and validated them by thermophoresis binding and protein-protein interaction assays. The results provide a proof-of-concept for using this strategy in the high-throughput search of improved drug-like peptide binders that block therapeutically relevant protein-protein interactions.

Furan warheads for covalent trapping of weak protein-protein interactions: cross-linking of thymosin ß4 to actin.

We describe furan as a triggerable 'warhead' for site-specific cross-linking using the actin and thymosin ß4 (Tß4)-complex as model of a weak and dynamic protein-protein interaction (PPI) with known 3D structure and with application potential in disease contexts. The identified cross-linked residues demonstrate that lysine is a target for the furan warhead. The presented in vitro validation of covalently acting 'furan-armed' Tß4-variants provides initial proof to further exploit furan-technology for covalent drug design targeting lysines.

The Pseudomonas aeruginosa substrate-binding protein Ttg2D functions as a general glycerophospholipid transporter across the periplasm.

In Pseudomonas aeruginosa, Ttg2D is the soluble periplasmic phospholipid-binding component of an ABC transport system thought to be involved in maintaining the asymmetry of the outer membrane. Here we use the crystallographic structure of Ttg2D at 2.5 Å resolution to reveal that this protein can accommodate four acyl chains. Analysis of the available structures of Ttg2D orthologs shows that they conform a new substrate-binding-protein structural cluster. Native and denaturing mass spectrometry experiments confirm that Ttg2D, produced both heterologously and homologously and isolated from the periplasm, can carry two diacyl glycerophospholipids as well as one cardiolipin. Binding is notably promiscuous, allowing the transport of various molecular species. In vitro binding assays coupled to native mass spectrometry show that binding of cardiolipin is spontaneous. Gene knockout experiments in P. aeruginosa multidrug-resistant strains reveal that the Ttg2 system is involved in low-level intrinsic resistance against certain antibiotics that use a lipid-mediated pathway to permeate through membranes.

Probing the Kinetic and Thermodynamic Fingerprints of Anti-EGF Nanobodies by Surface Plasmon Resonance.

Despite the widespread use of antibodies in clinical applications, the precise molecular mechanisms underlying antibody-antigen (Ab-Ag) interactions are often poorly understood. In this study, we exploit the technical features of a typical surface plasmon resonance (SPR) biosensor to dissect the kinetic and thermodynamic components that govern the binding of single-domain Ab or nanobodies to their target antigen, epidermal growth factor (EGF), a key oncogenic protein that is involved in tumour progression. By carefully tuning the experimental conditions and transforming the kinetic data into equilibrium constants, we reveal the complete picture of binding thermodynamics, including the energetics of the complex-formation transition state. This approach, performed using an experimentally simple and high-throughput setup, is expected to facilitate mechanistic studies of Ab-based therapies and, importantly, promote the rational development of new biological drugs with suitable properties.

Top-Down Proteomics Applied to Human Cerebrospinal Fluid.

Cerebrospinal fluid (CSF) is the fluid of choice to study pathologies and disorders of the central nervous system (CNS). Its composition, especially its proteins and peptides, holds the promise that it may reflect the pathological state of an individual. Traditionally, proteins and peptides in CSF have been analyzed using bottom-up proteomics technologies in the search of high proteome coverage. However, the limited protein sequence coverage of this technology means that information regarding post-translational modifications (PTMs) and alternative splice variants is lost. As an alternative technology, top-down proteomics offers low to medium proteome coverage, but high protein coverage enabling almost a full characterization of the proteins' primary structure. This allows us to precisely identify distinct molecular forms of proteins (proteoforms) as well as naturally occurring bioactive peptide fragments, which could be of critical biological relevance and would otherwise remain undetected with a classical proteomics approach.Here, we describe various strategies including sample preparation protocols, off-line intact protein prefractionation, and LC-MS/MS methods together with data analysis pipelines to analyze cerebrospinal fluid (CSF) by top-down proteomics. However, there is not a unique or standardized method and the selection of the top-down strategy will depend on the exact goal of the study. Here, we describe various top-down proteomics methods that enable rapid protein characterization and may be an excellent companion analytical workflow in the search for new protein biomarkers in neurodegenerative diseases.

Specific expression pattern of tissue cytokines analyzed through the Surface Acoustic Wave technique is associated with age-related spontaneous benign prostatic hyperplasia in rats.

The aim of the study reported herein was to evaluate the suitability of the Surface Acoustic Wave (SAW) technique as a possible diagnostic tool in benign prostatic hyperplasia (BPH). Moreover, for the first time, the BPH model was a totally physiological using naturally aged rats with spontaneous, age-related BPH instead of the pharmacologically induced models usually used. Eighteen male Wistar rats were distributed according to their age: 6 weeks (young), 12 weeks (adult) and 12 months (old) old. Prostate gland was removed and analyzed by mini-arrays, Western blotting (WB) and SAW techniques. Mini-arrays indicated that there were significant differences in the expression of 29/34 inflammation-related cytokines. WB was carried out to confirm the results after selection of 4 cytokines from which one showed no changes, namely PDGF-AA, and the other three, which significantly increase in older animals, were CD86, ß-NGF and VEGF. Notwithstanding, WB of old rats yielded confusing results due to an anomalous migration of proteins, dismissing this technique as an useful tool in these animals. Accurate results in old rats were uniquely obtained by using the SAW technique. Thus, SAW analysis showed that there were not differences among groups in the amount of PDGF-AA. On the contrary, SAW analysis showed that amounts of CD86, ß-NGF and VEGF in old rats were 2.0, 1.9 and 5.7-fold higher than that from young ones, respectively. These results indicate that SAW is a highly accurate technique for determining changes in the cytokines expression in BPH.

Toward a Novel Drug To Target the EGF-EGFR Interaction: Design of Metabolically Stable Bicyclic Peptides.

In cancer, proliferation of malignant cells is driven by overactivation of growth-signalling mechanisms, such as the epidermal growth factor receptor (EGFR) pathway. Despite its therapeutic relevance, the EGF-EGFR interaction has remained elusive to inhibition by synthetic molecules, mostly as a result of its large size and lack of binding pockets and cavities. Designed peptides, featuring cyclic motifs and other structural constraints, have the potential to modulate such challenging protein-protein interactions (PPIs). Herein, we present the structure-based design of a series of bicyclic constrained peptides that mimic an interface domain of EGFR and inhibit the EGF-EGFR interaction by targeting the smaller partner (i.e., EGF). This design process was guided by the integrated use of in silico methods and biophysical techniques, such as NMR spectroscopy and surface acoustic wave. The best analogues were able to reduce selectively the viability of EGFR+ human cancer cells. In addition to their efficacy, these bicyclic peptides are endowed with exceptional stability and metabolic resistance-two features that make them suitable candidates for in vivo applications.

Inhibitory properties of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) derivatives acting on glycogen metabolising enzymes.

Glycogen synthase (GS) and glycogen phosphorylase (GP) are the key enzymes that control, respectively, the synthesis and degradation of glycogen, a multi-branched glucose polymer that serves as a form of energy storage in bacteria, fungi and animals. An abnormal glycogen metabolism is associated with several human diseases. Thus, GS and GP constitute adequate pharmacological targets to modulate cellular glycogen levels by means of their selective inhibition. The compound 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) is a known potent inhibitor of GP. We studied the inhibitory effect of DAB, its enantiomer LAB, and 29 DAB derivatives on the activity of rat muscle glycogen phosphorylase (RMGP) and E. coli glycogen synthase (EcGS). The isoform 4 of sucrose synthase (SuSy4) from Solanum tuberosum L. was also included in the study for comparative purposes. Although these three enzymes possess highly conserved catalytic site architectures, the DAB derivatives analysed showed extremely diverse inhibitory potential. Subtle changes in the positions of crucial residues in their active sites are sufficient to discriminate among the structural differences of the tested inhibitors. For the two Leloir-type enzymes, EcGS and SuSy4, which use sugar nucleotides as donors, the inhibitory potency of the compounds analysed was synergistically enhanced by more than three orders of magnitude in the presence of ADP and UDP, respectively. Our results are consistent with a model in which these compounds bind to the subsite in the active centre of the enzymes that is normally occupied by the glucosyl residue which is transferred between donor and acceptor substrates. The ability to selectively inhibit the catalytic activity of the key enzymes of the glycogen metabolism may represent a new approach for the treatment of disorders of the glycogen metabolism.

Peptides Targeting EGF Block the EGF-EGFR Interaction.

Epidermal growth factor receptor (EGFR) is a key target in chemotherapy. Some drugs acting on the receptor are currently in use; however, drug resistance, which causes tumour relapse, calls for the discovery of alternative inhibitors. Using docking and receptor hotspot mimicry, we have designed novel peptides directed at EGF, the main growth factor ligand of EGFR. An array of biophysical techniques was used to characterise the structure and interaction of these ligands with the target protein. Both design methods identified peptides able to bind EGF, and the capacity of these peptides to inhibit the interaction between EGF and EGFR was demonstrated in two in vitro systems. Based on targeting the smaller companion of a protein-protein interaction, the new approach described herein can be envisaged as a parallel drug design strategy, and our compounds represent the first in a new class of binders that could serve as complementary compounds in potential multidrug cancer therapy.

Supramolecular Adducts of Cucurbit[7]uril and Amino Acids in the Gas Phase.

The complexation of the macrocyclic cavitand cucurbit[7]uril (Q7) with a series of amino acids (AA) with different side chains (Asp, Asn, Gln, Ser, Ala, Val, and Ile) is investigated by ESI-MS techniques. The 1:1 [Q7 + AA + 2H](2+) adducts are observed as the base peak when equimolar Q7:AA solutions are electrosprayed, whereas the 1:2 [Q7 + 2AA + 2H](2+) dications are dominant when an excess of the amino acid is used. A combination of ion mobility mass spectrometry (IM-MS) and DFT calculations of the 1:1 [Q7 + AA + 2H](2+) (AA = Tyr, Val, and Ser) adducts is also reported and proven to be unsuccessful at discriminating between exclusion or inclusion-type conformations in the gas phase. Collision induced dissociation (CID) revealed that the preferred dissociation pathways of the 1:1 [Q7 + AA + 2H](2+) dications are strongly influenced by the identity of the amino acid side chain, whereas ion molecule reactions towards N-butylmethylamine displayed a common reactivity pattern comprising AA displacement. Special emphasis is given on the differences between the gas-phase behavior of the supramolecular adducts with amino acids (AA = Asp, Asn, Gln, Ser, Ala, Val, and Ile) and those featuring basic (Lys and Arg) and aromatic (Tyr and Phe) side chains.

N-Lauroylation during the Expression of Recombinant N-Myristoylated Proteins: Implications and Solutions.

Incorporation of myristic acid onto the N terminus of a protein is a crucial modification that promotes membrane binding and correct localization of important components of signaling pathways. Recombinant expression of N-myristoylated proteins in Escherichia coli can be achieved by co-expressing yeast N-myristoyltransferase and supplementing the growth medium with myristic acid. However, undesired incorporation of the 12-carbon fatty acid lauric acid can also occur (leading to heterogeneous samples), especially when the available carbon sources are scarce, as it is the case in minimal medium for the expression of isotopically enriched samples. By applying this method to the brain acid soluble protein 1 and the 1-185 N-terminal region of c-Src, we show the significant, and protein-specific, differences in the membrane binding properties of lauroylated and myristoylated forms. We also present a robust strategy for obtaining lauryl-free samples of myristoylated proteins in both rich and minimal media.

Correction: Selective photoregulation of the activity of glycogen synthase and glycogen phosphorylase, two key enzymes in glycogen metabolism.

Correction for 'Selective photoregulation of the activity of glycogen synthase and glycogen phosphorylase, two key enzymes in glycogen metabolism' by Mireia Díaz-Lobo, et al., Org. Biomol. Chem., 2015, 13, 7282-7288.

Selective photoregulation of the activity of glycogen synthase and glycogen phosphorylase, two key enzymes in glycogen metabolism.

Glycogen is a polymer of α-1,4- and α-1,6-linked glucose units that provides a readily available source of energy in living organisms. Glycogen synthase (GS) and glycogen phosphorylase (GP) are the two enzymes that control, respectively, the synthesis and degradation of this polysaccharide and constitute adequate pharmacological targets to modulate cellular glycogen levels, by means of inhibition of their catalytic activity. Here we report on the synthesis and biological evaluation of a selective inhibitor that consists of an azobenzene moiety glycosidically linked to the anomeric carbon of a glucose molecule. In the ground state, the more stable (E)-isomer of the azobenzene glucoside had a slight inhibitory effect on rat muscle GP (RMGP, IC50 = 4.9 mM) and Escherichia coli GS (EcGS, IC50 = 1.6 mM). After irradiation and subsequent conversion to the (Z)-form, the inhibitory potency of the azobenzene glucoside did not significantly change for RMGP (IC50 = 2.4 mM), while its effect on EcGS increased 50-fold (IC50 = 32 µM). Sucrose synthase 4 from potatoes, a glycosyltransferase that does not operate on glycogen, was only slightly inhibited by the (E)-isomer (IC50 = 0.73 mM). These findings could be rationalized on the basis of kinetic and computer-aided docking analysis, which indicated that both isomers of the azobenzene glucoside mimic the EcGS acceptor substrate and exert their inhibitory effect by binding to the glycogen subsite in the active center of the enzyme. The ability to selectively photoregulate the catalytic activity of key enzymes of glycogen metabolism may represent a new approach for the treatment of glycogen metabolism disorders.

Lyase activity of glycogen synthase: Is an elimination/addition mechanism a possible reaction pathway for retaining glycosyltransferases?

Despite the biological relevance of glycosyltrasferases (GTs) and the many efforts devoted to this subject, the catalytic mechanism through which a subclass of this large family of enzymes, namely those that operate with net retention of the anomeric configuration, has not been fully established. Here, we show that in the absence of an acceptor, an archetypal retaining GT such as Pyrococcus abyssi glycogen synthase (PaGS) reacts with its glucosyl donor substrate, uridine 5'-diphosphoglucose (UDP-Glc), to produce the scission of the covalent bond between the terminal phosphate oxygen of UDP and the sugar ring. X-ray diffraction analysis of the PaGS/UDP-Glc complex shows no electronic density attributable to the UDP moiety, but establishes the presence in the active site of the enzyme of a glucose-like derivative that lacks the exocyclic oxygen attached to the anomeric carbon. Chemical derivatization followed by gas chromatography/mass spectrometry of the isolated glucose-like species allowed us to identify the molecule found in the catalytic site of PaGS as 1,5-anhydro-D-arabino-hex-1-enitol (AA) or its tautomeric form, 1,5-anhydro-D-fructose. These findings are consistent with a stepwise S(N) i-like mechanism as the modus operandi of retaining GTs, a mechanism that involves the discrete existence of an oxocarbenium intermediate. Even in the absence of a glucosyl acceptor, glycogen synthase (GS) promotes the formation of the cationic intermediate, which, by eliminating the proton of the adjacent C2 carbon atom, yields AA. Alternatively, these observations could be interpreted assuming that AA is a true intermediate in the reaction pathway of GS and that this enzyme operates through an elimination/addition mechanism.