The Complete Assessment of Small Molecule and Peptidomimetic Inhibitors of Sortase A Towards Antivirulence Treatment.

This review covers the most recent advances in the development of inhibitors for the bacterial enzyme sortase A (SrtA). Sortase A (SrtA) is a critical virulence factor, present ubiquitously in Gram-positive bacteria of which many are pathogenic. Sortases are key enzymes regulating bacterial adherence to host cells, by anchoring extracellular matrix-binding proteins to the bacterial outer cell wall. By targeting virulence factors, effective treatment can be achieved, without inducing antibiotic resistance to the treatment. This is a potentially more sustainable, long-term approach to treating bacterial infections, including ones that display multiple resistance to current therapeutics. There are many promising approaches available for SrtA inhibition, some of which have the potential to advance into further clinical development, with peptidomimetic and in vivo active small molecules being among the most promising. There are currently no approved drugs on the market targeting SrtA, despite its promise, adding to the relevance of this review article, as it extends to the pharmaceutical industry additionally to academic researchers.

Mimicking Nonribosomal Peptides from the Marine Actinomycete Streptomyces sp. H-KF8 Leads to Antimicrobial Peptides.

Microorganisms within the marine environment have been shown to be very effective sources of naturally produced antimicrobial peptides (AMPs). Several nonribosomal peptides were identified based on genome mining predictions of Streptomyces sp. H-KF8, a marine Actinomycetota isolated from a remote Northern Chilean Patagonian fjord. Based on these predictions, a series of eight peptides, including cyclic peptides, were designed and chemically synthesized. Six of these peptides showed antimicrobial activity. Mode of action studies suggest that two of these peptides potentially act on the cell membrane via a novel mechanism allowing the passage of small ions, resulting in the dissipation of the membrane potential. This study shows that though structurally similar peptides, determined by NMR spectroscopy, the incorporation of small sequence mutations results in a dramatic influence on their bioactivity including mode of action. The qualified hit sequence can serve as a basis for more potent AMPs in future studies.

Substrate-derived Sortase A inhibitors: targeting an essential virulence factor of Gram-positive pathogenic bacteria.

The bacterial transpeptidase Sortase A (SrtA) is a surface enzyme of Gram-positive pathogenic bacteria. It has been shown to be an essential virulence factor for the establishment of various bacterial infections, including septic arthritis. However, the development of potent Sortase A inhibitors remains an unmet challenge. Sortase A relies on a five amino acid sorting signal (LPXTG), by which it recognizes its natural target. We report the synthesis of a series of peptidomimetic inhibitors of Sortase A based on the sorting signal, supported by computational binding analysis. By employing a FRET-compatible substrate, our inhibitors were assayed in vitro. Among our panel, we identified several promising inhibitors with IC50 values below 200 µM, with our strongest inhibitor - LPRDSar - having an IC50 of 18.9 µM. Furthermore, it was discovered that three of our compounds show an effect on growth and biofilm inhibition of pathogenic Staphylococcus aureus, with the inclusion of a phenyl ring seemingly key to this effect. The most promising compound in our panel, BzLPRDSar, could inhibit biofilm formation at concentrations as low as 32 µg mL-1, manifesting it as a potential future drug lead. This could lead to treatments for MRSA infections in clinics and diseases such as septic arthritis, which has been directly linked with SrtA.

Protein cohabitation: long-term immunoglobulin G storage at room temperature.

Long-term functional storage of therapeutic proteins at room temperature has been an eternal challenge. Inspired by the cellular cooperativity of proteins, we have taken a step forward to address this challenge by cohabitating Immunoglobulin G (IgG1) with a food protein gelatin in the solid-state at room temperature. Interestingly, IgG1 remained functionally active for a record 14 months revealed from the western-blot assay. Further quantification by HP-LC analysis showed 100% structural integrity of IgG1 with no degradation in the gelatin matrix during this period. The developed formulation has a direct application in oral medical nutrition therapy to cure gastrointestinal microbial infections. Also the strategy provides a robust energy economic alternative to the protein engineering methods for long-term functional storage of therapeutic proteins at room temperature.

CA10 regulates neurexin heparan sulfate addition via a direct binding in the secretory pathway.

Neurexins are presynaptic adhesion molecules that shape the molecular composition of synapses. Diversification of neurexins in numerous isoforms is believed to confer synapse-specific properties by engaging with distinct ligands. For example, a subset of neurexin molecules carry a heparan sulfate (HS) glycosaminoglycan that controls ligand binding, but how this post-translational modification is controlled is not known. Here, we observe that CA10, a ligand to neurexin in the secretory pathway, regulates neurexin-HS formation. CA10 is exclusively found on non-HS neurexin and CA10 expressed in neurons is sufficient to suppress HS addition and attenuate ligand binding and synapse formation induced by ligands known to recruit HS. This effect is mediated by a direct interaction in the secretory pathway that blocks the primary step of HS biosynthesis: xylosylation of the serine residue. NMR reveals that CA10 engages residues on either side of the serine that can be HS-modified, suggesting that CA10 sterically blocks xylosyltransferase access in Golgi. These results suggest a mechanism for the regulation of HS on neurexins and exemplify a new mechanism to regulate site-specific glycosylations.

Native Chemical Ligation of Highly Hydrophobic Peptides in Ionic Liquid-Containing Media.

The chemical synthesis of a highly hydrophobic membrane-associated peptide by native chemical ligation (NCL) in an ionic liquid (IL) [C2mim][OAc]/buffer mixture was achieved by employing peptide concentrations up to 11 mM. NCL was studied at different pH and water content and compared to several "gold-standard" ligation protocols. The optimized reaction protocol for the NCL in IL required the addition of 40% water and pH adjustment to 7.0-7.5, resulting in ligation yields of up to 80-95% within 1 to 4 h. This new ligation protocol is generally applicable and outperforms current "gold-standard" NCL methods.

Ultrasensitive and Selective Copper(II) Detection: Introducing a Bioinspired and Robust Sensor.

A nanopore-based CuII -sensing system is reported that allows for an ultrasensitive and selective detection of CuII with the possibility for a broad range of applications, for example in medical diagnostics. A fluorescent ATCUN-like peptide 5/6-FAM-Dap-ß-Ala-His is employed to selectively bind CuII ions in the presence of NiII and ZnII and was crafted into ion track-etched nanopores. Upon CuII binding the fluorescence of the peptide sensor is quenched, permitting the detection of CuII in solution. The ion transport characteristics of peptide-modified nanopore are shown to be extremely sensitive and selective towards CuII allowing to sense femtomolar CuII concentrations in human urine mimics. Washing with EDTA fully restores the CuII -binding properties of the sensor, enabling multiple repetitive measurements. The robustness of the system clearly has the potential to be further developed into an easy-to-use, lab-on-chip CuII -sensing device, which will be of great importance for bedside diagnosis and monitor of CuII levels in patients with copper-dysfunctional homeostasis.

Structural and Dynamical Basis of G Protein Inhibition by YM-254890 and FR900359: An Inhibitor in Action.

Specific inhibition of G proteins holds a great pharmacological promise to, e.g., target oncogenic Gq/11 proteins and can be achieved by the two natural products FR900359 (FR) and YM-254890 (YM). Unfortunately, recent rational-design-based approaches to address G proteins other than Gq/11/14 subtypes were not successful mainly due to the conformational complexity of these new modalities-like compounds. Here, we report the water-derived NMR structure of YM, which strongly differs from the conformation of Gq-bound YM as found in the crystal structure. Reanalysis of the crystal structure suggests that the water-derived NMR structure of YM also represents a valid solution of the electron density. Extensive molecular dynamic simulations unveiled much higher binding affinities of the water-derived NMR structure compared to the original YM conformation of pdb 3ah8 . Employing a in-silico-designed, fast activating G protein conformation molecular dynamics data ultimately show how the inhibitor impairs the domain motion of the G protein necessary to hinder nucleotide exchange.

Discovery and development of substituted thiadiazoles as inhibitors of Staphylococcus aureus Sortase A.

High-throughput screening of small-molecule libraries has led to the identification of thiadiazoles as a new class of inhibitors against Staphylococcus aureus sortase A (SrtA). N-(5-((4-nitrobenzyl)thio)-1,3,4-thiadiazol-2-yl)nicotinamide (IC50 = 3.8 µM) was identified as a potent inhibitor of SrtA after synthetic modification of hit compounds. Additional ligands developed in this study displayed affinities in the low micromolar range without affecting bacterial growth in vitro. The study also suggest a new mode of action through covalent binding to the active site cysteine.

Effect of Conformational Diversity on the Bioactivity of µ-Conotoxin PIIIA Disulfide Isomers.

Cyclic µ-conotoxin PIIIA, a potent blocker of skeletal muscle voltage-gated sodium channel NaV1.4, is a 22mer peptide stabilized by three disulfide bonds. Combining electrophysiological measurements with molecular docking and dynamic simulations based on NMR solution structures, we investigated the 15 possible 3-disulfide-bonded isomers of µ-PIIIA to relate their blocking activity at NaV1.4 to their disulfide connectivity. In addition, three µ-PIIIA mutants derived from the native disulfide isomer, in which one of the disulfide bonds was omitted (C4-16, C5-C21, C11-C22), were generated using a targeted protecting group strategy and tested using the aforementioned methods. The 3-disulfide-bonded isomers had a range of different conformational stabilities, with highly unstructured, flexible conformations with low or no channel-blocking activity, while more constrained molecules preserved 30% to 50% of the native isomer's activity. This emphasizes the importance and direct link between correct fold and function. The elimination of one disulfide bond resulted in a significant loss of blocking activity at NaV1.4, highlighting the importance of the 3-disulfide-bonded architecture for µ-PIIIA. µ-PIIIA bioactivity is governed by a subtle interplay between an optimally folded structure resulting from a specific disulfide connectivity and the electrostatic potential of the conformational ensemble.

In Silico Analysis of the Subtype Selective Blockage of KCNA Ion Channels through the µ-Conotoxins PIIIA, SIIIA, and GIIIA.

Understanding subtype specific ion channel pore blockage by natural peptide-based toxins is crucial for developing such compounds into promising drug candidates. Herein, docking and molecular dynamics simulations were employed in order to understand the dynamics and binding states of the µ-conotoxins, PIIIA, SIIIA, and GIIIA, at the voltage-gated potassium channels of the KV1 family, and they were correlated with their experimental activities recently reported by Leipold et al. Their different activities can only adequately be understood when dynamic information about the toxin-channel systems is available. For all of the channel-bound toxins investigated herein, a certain conformational flexibility was observed during the molecular dynamic simulations, which corresponds to their bioactivity. Our data suggest a similar binding mode of µ-PIIIA at KV1.6 and KV1.1, in which a plethora of hydrogen bonds are formed by the Arg and Lys residues within the α-helical core region of µ-PIIIA, with the central pore residues of the channel. Furthermore, the contribution of the K+ channel's outer and inner pore loops with respect to the toxin binding. and how the subtype specificity is induced, were proposed.

Rational design of a heterotrimeric G protein α subunit with artificial inhibitor sensitivity.

Transmembrane signals initiated by a range of extracellular stimuli converge on members of the Gq family of heterotrimeric G proteins, which relay these signals in target cells. Gq family G proteins comprise Gq, G11, G14, and G16, which upon activation mediate their cellular effects via inositol lipid-dependent and -independent signaling to control fundamental processes in mammalian physiology. To date, highly specific inhibition of Gq/11/14 signaling can be achieved only with FR900359 (FR) and YM-254890 (YM), two naturally occurring cyclic depsipeptides. To further development of FR or YM mimics for other Gα subunits, we here set out to rationally design Gα16 proteins with artificial FR/YM sensitivity by introducing an engineered depsipeptide-binding site. Thereby we permit control of G16 function through ligands that are inactive on the WT protein. Using CRISPR/Cas9-generated Gαq/Gα11-null cells and loss- and gain-of-function mutagenesis along with label-free whole-cell biosensing, we determined the molecular coordinates for FR/YM inhibition of Gq and transplanted these to FR/YM-insensitive G16. Intriguingly, despite having close structural similarity, FR and YM yielded biologically distinct activities: it was more difficult to perturb Gq inhibition by FR and easier to install FR inhibition onto G16 than perturb or install inhibition with YM. A unique hydrophobic network utilized by FR accounted for these unexpected discrepancies. Our results suggest that non-Gq/11/14 proteins should be amenable to inhibition by FR scaffold-based inhibitors, provided that these inhibitors mimic the interaction of FR with Gα proteins harboring engineered FR-binding sites.

Ultrafast Single-Scan 2D†NMR Spectroscopic Detection of a PHIP-Hyperpolarized Protease Inhibitor.

Two-dimensional NMR spectroscopy is one of the most important spectroscopic tools for the investigation of biological macromolecules. However, due to the low sensitivity of NMR spectroscopy, it takes usually from several minutes to many hours to record such spectra. Here, the possibility of detecting a bioactive derivative of the sunflower trypsin inhibitor-1 (SFTI-1), a tetradecapeptide, by combining parahydrogen-induced polarization (PHIP) and ultrafast 2D NMR spectroscopy is shown. The PHIP activity of the inhibitor was achieved by labeling with O-propargyl-l-tyrosine. In 1D PHIP experiments a signal enhancement of a factor of approximately 1200 compared to standard NMR was found. This enhancement permits measurement of 2D NMR correlation spectra of low-concentrated SFTI-1 in less than 10 seconds, employing ultrafast single-scan 2D NMR detection. As experimental examples PHIP-assisted ultrafast single-scan TOCSY spectra of SFTI-1 are shown.

NiII Complex Formation and Protonation States at the Active Site of a Nickel Superoxide Dismutase-Derived Metallopeptide: Implications for the Mechanism of Superoxide Degradation.

A small, catalytically active metallopeptide (Nim6 SOD, m6 SOD=ACDLAC), which was derived from the nickel superoxide dismutase (NiSOD) active site was employed to study the mechanism of superoxide degradation, especially focusing on the protonation states of the NiII donor atoms, the proton source, and the role of the N-terminal proton(s). Therefore, the NiII -metallopeptide was studied at various pHs and temperatures using UV/Vis and NMR spectroscopy. These studies indicate a strong reduction of the pKa of the NiII -ligating donor atoms, resulting in a fully deprotonated NiII active-site environment. Furthermore, no titratable proton could be observed within a pH ranging from 6.5 to 10.5. This rules out a recently discussed adiabatic proton tunneling-like hydrogen-atom transfer process for the metallopeptides, not found in the native enzyme. Furthermore, variable-temperature 1 H NMR measurements uncovered an extended hydrogen-bond network within the NiII active site of the metallopeptide similar to the enzyme. With respect to the deprotonated NiII active site, the residual N-terminal proton, which is a prerequisite for catalytic activity, cannot act as proton source. Most likely, it stabilizes the NiII -coordinated substrate in an end-on fashion, thus allowing for an inner-sphere electron transfer. Lastly, and unlike the enzyme, the catalytic rate constant of superoxide degradation by the metallopeptides was determined to be strongly pH dependent, suggesting bulk water to be directly involved in proton donation, which in turn strongly suggests the N-terminal histidine to be the respective proton donor in the enzyme.

Deciphering Specificity Determinants for FR900359-Derived Gq α Inhibitors Based on Computational and Structure-Activity Studies.

Direct targeting of intracellular Gα subunits of G protein-coupled receptors by chemical tools is a challenging task in current pharmacological studies and in the development of novel therapeutic approaches. In this study we analyzed novel FR900359-based analogs from natural sources, synthetic cyclic peptides, as well as all so-far known Gq α inhibitors in a comprehensive study to devise a strategy for the elucidation of characteristics that determine interactions with and inhibition of Gq in the specific FR/YM-binding pocket. Using 2D NMR spectroscopy and molecular docking we identified unique features in the macrocyclic structures responsible for binding to the target protein correlating with inhibitory activity. While all novel compounds were devoid of effects on Gi and Gs proteins, no inhibitor surpassed the biological activity of FR. This raises the question of whether depsipeptides such as FR already represent valuable chemical tools for specific inhibition of Gq and, at the same time, are suitable natural lead structures for the development of novel compounds to target Gα subunits other than Gq .

Conformational µ-Conotoxin PIIIA Isomers Revisited: Impact of Cysteine Pairing on Disulfide-Bond Assignment and Structure Elucidation.

Peptides and proteins carrying high numbers of cysteines can adopt various 3D structures depending on their disulfide connectivities. The unambiguous verification of such conformational isomers with more than two disulfide bonds is extremely challenging, and experimental strategies for their unequivocal structural analysis are largely lacking. We synthesized all 15 possible isomers of the 22mer conopeptide µ-PIIIA and applied 2D NMR spectroscopy and MS/MS for the elucidation of its structure. This study provides intriguing insights in how the disulfide connectivity alters the global fold of a toxin. We also show that analysis procedures involving comprehensive combinations of conventional methods are required for the unambiguous assignment of disulfides in cysteine-rich peptides and proteins and that standard compounds are crucially needed for the structural analysis of such complex molecules.

New insights into the mechanism of nickel superoxide degradation from studies of model peptides.

A series of small, catalytically active metallopeptides, which were derived from the nickel superoxide dismutase (NiSOD) active site were employed to study the mechanism of superoxide degradation especially focusing on the role of the axial imidazole ligand. In the literature, there are contradicting propositions about the catalytic importance of the N-terminal histidine. Therefore, we studied the stability and activity of a set of eight NiSOD model peptides, which represent the major model systems discussed in the literature to date, yet differing in their length and their Ni-coordination. UV-Vis-coupled stopped-flow kinetic measurements and mass spectrometry analysis unveiled their high oxidation sensitivity in the presence of oxygen and superoxide resulting into a much faster Ni(II)-peptide degradation for the amine/amide Ni(II) coordination than for the catalytically inactive bis-amidate Ni(II) coordination. With respect to these results we determined the catalytic activities for all NiSOD mimics studied herein, which turned out to be in almost the same range of about 2 × 106 M-1 s-1. From these experiments, we concluded that the amine/amide Ni(II) coordination is clearly the key factor for catalytic activity. Finally, we were able to clarify the role of the N-terminal histidine and to resolve the contradictory literature propositions, reported in previous studies.

Reactions of Sulfur-Containing Organic Compounds and Peptides in 1-Ethyl-3-methyl-imidazolium Acetate.

The neat ionic liquid (IL) [C2mim][OAc] is not just capable of dissolving thiol- and disulfide-containing compounds, but is able to chemically react with them without addition of any catalytic reagent. Through the analysis of four small organic molecules and a cysteine-containing peptide we could postulate a general reaction mechanism. Here, the imidazolium-carbenes preferentially react with the disulfide bond, but not thiol group. Moreover, the imidazole moiety was found to abstract the sulfur atom from the cysteine residue, providing an alternative way to transform Cys residues, which were artificially inserted into a peptide sequence in order to perform native chemical ligation (NCL) of two peptide fragments. Finally, the chemical reaction of [C2mim][OAc] with a cysteine-containing biomolecules can be tuned or even suppressed through the addition of at least 30% of water to the reaction mixture.

Solid-State NMR Investigation of the Conformation, Proton Conduction, and Hydration of the Influenza B Virus M2 Transmembrane Proton Channel.

Together with the influenza A virus, influenza B virus causes seasonal flu epidemics. The M2 protein of influenza B (BM2) forms a tetrameric proton-conducting channel that is important for the virus lifecycle. BM2 shares little sequence homology with AM2, except for a conserved HxxxW motif in the transmembrane (TM) domain. Unlike AM2, no antiviral drugs have been developed to block the BM2 channel. To elucidate the proton-conduction mechanism of BM2 and to facilitate the development of BM2 inhibitors, we have employed solid-state NMR spectroscopy to investigate the conformation, dynamics, and hydration of the BM2 TM domain in lipid bilayers. BM2 adopts an α-helical conformation in lipid membranes. At physiological temperature and low pH, the proton-selective residue, His19, shows relatively narrow (15)N chemical exchange peaks for the imidazole nitrogens, indicating fast proton shuttling that interconverts cationic and neutral histidines. Importantly, pH-dependent (15)N chemical shifts indicate that His19 retains the neutral population to much lower pH than His37 in AM2, indicating larger acid-dissociation constants or lower pKa's. We attribute these dynamical and equilibrium differences to the presence of a second titratable histidine, His27, which may increase the proton-dissociation rate of His19. Two-dimensional (1)H-(13)C correlation spectra probing water (1)H polarization transfer to the peptide indicates that the BM2 channel becomes much more hydrated at low pH than at high pH, particularly at Ser12, indicating that the pore-facing serine residues in BM2 mediate proton relay to the proton-selective histidine.