RESUMEN
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 inâ 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.
Asunto(s)
Aminoaciltransferasas , Antibacterianos , Proteínas Bacterianas , Cisteína Endopeptidasas , Peptidomiméticos , Bibliotecas de Moléculas Pequeñas , Aminoaciltransferasas/antagonistas & inhibidores , Aminoaciltransferasas/metabolismo , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/química , Peptidomiméticos/química , Peptidomiméticos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Antibacterianos/química , Antibacterianos/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Humanos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Bacterias Grampositivas/efectos de los fármacosRESUMEN
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.
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Proteínas del Tejido Nervioso , Moléculas de Adhesión de Célula Nerviosa , Proteínas de Unión al Calcio/metabolismo , Heparitina Sulfato/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Vías Secretoras , Sinapsis/metabolismoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Depsipéptidos/farmacología , Inhibidores Enzimáticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP , Péptidos Cíclicos/farmacología , Ingeniería de Proteínas , Animales , Sistemas CRISPR-Cas , Subunidades alfa de la Proteína de Unión al GTP/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , RatonesRESUMEN
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.
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Cobre/análisis , Iones/química , Péptidos/química , Cobre/química , Cobre/metabolismo , Fluorescencia , Humanos , Péptidos/metabolismoRESUMEN
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 2Dâ NMR spectroscopy is shown. The PHIP activity of the inhibitor was achieved by labeling with O-propargyl-l-tyrosine. In 1Dâ PHIP experiments a signal enhancement of a factor of approximately 1200 compared to standard NMR was found. This enhancement permits measurement of 2Dâ NMR correlation spectra of low-concentrated SFTI-1 in less than 10â seconds, employing ultrafast single-scan 2Dâ NMR detection. As experimental examples PHIP-assisted ultrafast single-scan TOCSY spectra of SFTI-1 are shown.
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Imidazoles/química , Inhibidores de Proteasas/análisis , Algoritmos , Espectroscopía de Resonancia Magnética/métodos , Estructura Molecular , Péptidos Cíclicos/análisis , Tirosina/análogos & derivados , Tirosina/químicaRESUMEN
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.
Asunto(s)
Depsipéptidos/farmacología , Proteínas de Unión al GTP/antagonistas & inhibidores , Péptidos Cíclicos/farmacología , Modelos Moleculares , Simulación de Dinámica Molecular , Estructura Molecular , Conformación ProteicaRESUMEN
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 (IC50â¯=â¯3.8⯵M) 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.
Asunto(s)
Aminoaciltransferasas/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Staphylococcus aureus/enzimología , Tiadiazoles/farmacología , Aminoaciltransferasas/química , Antibacterianos/síntesis química , Antibacterianos/metabolismo , Proteínas Bacterianas/química , Dominio Catalítico , Cisteína Endopeptidasas/química , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/metabolismo , Descubrimiento de Drogas , Escherichia coli/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-Actividad , Tiadiazoles/síntesis química , Tiadiazoles/metabolismoRESUMEN
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.
Asunto(s)
Conotoxinas/farmacología , Simulación de Dinámica Molecular , Canales de Potasio de la Superfamilia Shaker/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Conotoxinas/química , Unión Proteica , Homología de Secuencia de Aminoácido , Canales de Potasio de la Superfamilia Shaker/química , Canales de Potasio de la Superfamilia Shaker/metabolismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Conotoxinas/farmacocinética , Canal de Sodio Activado por Voltaje NAV1.4/química , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Conotoxinas/química , Disulfuros/química , Isomerismo , Simulación del Acoplamiento Molecular , Conformación Proteica , Electricidad Estática , Relación Estructura-Actividad , Bloqueadores del Canal de Sodio Activado por Voltaje/químicaRESUMEN
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.
RESUMEN
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 Hâ 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.
RESUMEN
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.
RESUMEN
In dicotyledonous plants, xyloglucan (XyG) is the most abundant hemicellulose of the primary cell wall. The enzymes involved in XyG biosynthesis have been identified through reverse-genetics and activity was characterized by heterologous expression. Currently, there is no information on the atomic structures or amino acids involved in activity or substrate binding of any of the Golgi-localized XyG biosynthetic enzymes. A homology model of the xyloglucan xylosyltransferase 2 (XXT2) catalytic domain was built on the basis of the crystal structure of A64Rp. Molecular dynamics simulations revealed that the homology model retains the glycosyltransferase (GT)-A fold of the template structure used to build the homology model indicating that XXT2 likely has a GT-A fold. According to the XXT2 homology model, six amino acids (Phe204, Lys207, Asp228, Ser229, Asp230, His378) were selected and their contribution in catalytic activity was investigated. Site-directed mutagenesis studies show that Asp228, Asp230 and His378 are critical for XXT2 activity and are predicted to be involved in coordination of manganese ion. Lys207 was also found to be critical for protein activity and the homology model indicates a critical role in substrate binding. Additionally, Phe204 mutants have less of an impact on XXT2 activity with the largest effect when replaced with a polar residue. This is the first study that investigates the amino acids involved in substrate binding of the XyG-synthesizing xylosyltransferases and contributes to the understanding of the mechanisms of polysaccharide-synthesizing GTs and XyG biosynthesis.
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Aminoácidos/química , Pared Celular/química , Pentosiltransferasa/química , Conformación Proteica , Secuencia de Aminoácidos/genética , Aminoácidos/genética , Arabidopsis/química , Arabidopsis/genética , Sitios de Unión , Dominio Catalítico , Pared Celular/enzimología , Glucanos/biosíntesis , Glucanos/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Pentosiltransferasa/genética , Unión Proteica , Homología Estructural de Proteína , Especificidad por Sustrato , Xilanos/biosíntesis , Xilanos/químicaRESUMEN
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.
Asunto(s)
Membrana Celular/metabolismo , Virus de la Influenza B , Resonancia Magnética Nuclear Biomolecular , Protones , Proteínas de la Matriz Viral/química , Agua/metabolismo , Frío , Histidina/metabolismo , Concentración de Iones de Hidrógeno , Modelos Moleculares , Conformación Proteica , Proteínas de la Matriz Viral/metabolismoRESUMEN
The influenza M2 protein forms an acid-activated tetrameric proton channel important for the virus lifecycle. Residue His37 in the transmembrane domain is responsible for channel activation and proton selectivity. While the structure and dynamics of His37 have been well studied in TM peptide constructs, it has not been investigated in the presence of the full cytoplasmic domain, which increases the proton conductivity by 2-fold compared to the TM peptide. We report here (13)C and (15)N chemical shifts of His37 in the cytoplasmic-containing M2(21-97) and show that cationic histidines are already present at neutral pH, in contrast to the TM peptide, indicating that the cytoplasmic domain shifts the protonation equilibria. Quantification of the imidazole (15)N intensities yielded two resolved proton dissociation constants (pKa's) of 7.1 and 5.4, which differ from the TM result but resemble the M2(18-60) result, suggesting cooperative proton binding. The average His37 pKa is higher for M2(21-97) than for the shorter constructs. We attribute this higher pKa to direct and indirect effects of the cytoplasmic domain, which is rich in acidic residues. 2D (13)C-(13)C correlation spectra reveal seven His37 Cα-Cß cross peaks at different pH, some of which are unique to the cytoplasmic-containing M2 and correspond to more ideal α-helical conformations. Based on the pH at which these chemical shifts appear and their side chain structures, we assign these conformations to His37 in differently charged tetramers. Thus, the cytoplasmic domain facilitates proton conduction through the transmembrane pore by modifying the His37-water proton exchange equilibria and the His37 backbone conformational distribution.
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Histidina/química , Protones , Proteínas de la Matriz Viral/química , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética/normas , Conformación de Ácido Nucleico , Estándares de Referencia , Proteínas de la Matriz Viral/biosíntesis , Proteínas de la Matriz Viral/aislamiento & purificaciónRESUMEN
A series of novel bioactive derivatives of the sunflower trypsin inhibitor-1 (SFTI-1) suitable for hyperpolarization by parahydrogen-induced polarization (PHIP) was developed. The PHIP activity was achieved by labeling with L-propargylglycine, O-propargyl-L-tyrosine, or 4-pentynoic acid. (1) Hâ NMR signal enhancements (SE) of up to a factor of 70 were achieved in aqueous solution. We found that an isolated spatial location of the triple bond within the respective label and its accessibility for the hydrogenation catalyst are essential factors for the degree of signal enhancement.
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Alquinos/química , Ácidos Grasos Insaturados/química , Glicina/análogos & derivados , Espectroscopía de Resonancia Magnética/métodos , Péptidos Cíclicos/química , Tirosina/análogos & derivados , Catálisis , Glicina/química , Hidrogenación , Modelos Moleculares , Estructura Molecular , Protones , Tirosina/químicaRESUMEN
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.
Asunto(s)
Actinobacteria , Streptomyces , Péptidos Antimicrobianos , Streptomyces/genética , Streptomyces/química , Péptidos/farmacología , Péptidos/metabolismo , Péptidos Cíclicos/químicaRESUMEN
The M2 protein of influenza A viruses forms a tetrameric proton channel that is targeted by the amantadine class of antiviral drugs. A S31N mutation in the transmembrane (TM) domain of the protein has caused widespread amantadine resistance in most of the currently circulating flu viruses. Recently, a new family of compounds based on amantadine- and aryl-substituted isoxazole were discovered to inhibit the S31N channel activity and reduce replication of S31N-harboring viruses. We now use solid-state NMR spectroscopy to investigate the effects of one of these isoxazole compounds, WJ352, on the conformation of the S31N TM segment and the dynamics of the proton-selective residue, His37. Chemical shift perturbations show that WJ352 changes the conformational equilibrium of multiple TM residues, with the maximal perturbation occurring at the crucial Asn31. (13)C-(2)H distance measurements and (1)H-(1)H NOE cross peaks indicate that the adamantane moiety of the drug is bound in the spacious pore between Asn31 and Gly34 while the phenyl tail is located near Val27. Thus, the polar amine points to the channel exterior rather than to His37, in contrast to amantadine and rimantadine in the wild-type channel, suggesting that the drug is significantly stabilized by hydrophobic interactions between the adamantane and the TM peptide. (15)N and (13)C chemical shifts indicate that at low pH, His37 undergoes fast exchange among the τ tautomer, the π tautomer, and the cationic state due to proton transfer with water. The exchange rate is higher than the wild-type channel, consistent with the larger single-channel conductance of the mutant. Drug binding at acidic pH largely suppresses this exchange, reverting the histidines to a similar charge distribution as that of the high-pH closed state.
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Amantadina/farmacología , Canales Iónicos/antagonistas & inhibidores , Isoxazoles/farmacología , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Proteínas de la Matriz Viral/antagonistas & inhibidores , Amantadina/química , Canales Iónicos/química , Canales Iónicos/metabolismo , Isoxazoles/química , Modelos Moleculares , Estructura Molecular , Mutación , Conformación Proteica/efectos de los fármacos , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/metabolismoRESUMEN
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.