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1.
Nat Commun ; 15(1): 4359, 2024 May 22.
Article in English | MEDLINE | ID: mdl-38777835

ABSTRACT

Cystine-knot peptides (CKPs) are naturally occurring peptides that exhibit exceptional chemical and proteolytic stability. We leveraged the CKP carboxypeptidase A1 inhibitor as a scaffold to construct phage-displayed CKP libraries and subsequently screened these collections against HTRA1, a trimeric serine protease implicated in age-related macular degeneration and osteoarthritis. The initial hits were optimized by using affinity maturation strategies to yield highly selective and potent picomolar inhibitors of HTRA1. Crystal structures, coupled with biochemical studies, reveal that the CKPs do not interact in a substrate-like manner but bind to a cryptic pocket at the S1' site region of HTRA1 and abolish catalysis by stabilizing a non-competent active site conformation. The opening and closing of this cryptic pocket is controlled by the gatekeeper residue V221, and its movement is facilitated by the absence of a constraining disulfide bond that is typically present in trypsin fold serine proteases, thereby explaining the remarkable selectivity of the CKPs. Our findings reveal an intriguing mechanism for modulating the activity of HTRA1, and highlight the utility of CKP-based phage display platforms in uncovering potent and selective inhibitors against challenging therapeutic targets.


Subject(s)
Catalytic Domain , High-Temperature Requirement A Serine Peptidase 1 , Peptides , High-Temperature Requirement A Serine Peptidase 1/metabolism , High-Temperature Requirement A Serine Peptidase 1/genetics , Humans , Peptides/chemistry , Peptides/metabolism , Peptides/pharmacology , Peptide Library , Crystallography, X-Ray , Protein Binding , Cystine/chemistry , Cystine/metabolism , Models, Molecular
2.
Anal Chem ; 96(16): 6459-6466, 2024 04 23.
Article in English | MEDLINE | ID: mdl-38592893

ABSTRACT

Cysteine (Cys) and its oxidized form, cystine (Cys2), play crucial roles in biological systems and have considerable applications in cell culture. However, Cys in cell culture media is easily oxidized to Cys2, leading to solubility issues. Traditional analytical methods struggle to maintain the oxidation states of Cys and Cys2 during analysis, posing a significant challenge to accurately measuring and controlling these compounds. To effectively control the Cys and Cys2 levels, a rapid and accurate analytical method is required. Here, we screened derivatizing reagents that can react with Cys even under acidic conditions to realize a novel analytical method for simultaneously determining Cys and Cys2 levels. Diethyl 2-methylenemalonate (EMM) was found to possess the desired traits. EMM, characterized by its dual electron-withdrawing attributes, allowed for a rapid reaction with Cys under acidic conditions, preserving intact information for understanding the functions of target compounds. Combined with LC-MS/MS and an internal standard, this method provided high analytical accuracy in a short analytical time of 9 min. Using the developed method, the rapid oxidation of Cys in cell culture media was observed with the headspace of the storage container considerably influencing Cys oxidation and Cys2 precipitation rates. The developed method enabled the direct and simplified analysis of Cys behavior in practical media samples and could be used in formulating new media compositions, ensuring quality assurance, and real-time analysis of Cys and Cys2 in cell culture supernatants. This novel approach holds the potential to further enhance the media performance by enabling the timely optimal addition of Cys.


Subject(s)
Culture Media , Cysteine , Cystine , Sulfhydryl Compounds , Tandem Mass Spectrometry , Chromatography, Liquid/methods , Click Chemistry , Culture Media/chemistry , Cysteine/chemistry , Cysteine/analysis , Cystine/chemistry , Cystine/analogs & derivatives , Cystine/analysis , Liquid Chromatography-Mass Spectrometry , Malonates/chemistry , Oxidation-Reduction , Sulfhydryl Compounds/chemistry , Sulfhydryl Compounds/analysis , Tandem Mass Spectrometry/methods
3.
Analyst ; 149(11): 3108-3114, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38639050

ABSTRACT

Here, we report a proof-of-concept resistive pulse method for analyzing chiral amino acids utilizing metal-amino acid crystallization differences. This method involves introducing an amino acid sample solution into a micropipette through a pressure-driven flow. The sample then mixes with a metal ion solution inside the pipette, forming metal-amino acid crystals. The crystal size depends on the enantiomeric excess (x) of chiral amino acid samples. Large x values lead to large crystals. The crystal size difference is then reflected in the resistive pulse size as they block the ionic transport in a micropipette to different extents. We used Cd-cystine crystallization as a model system and found approximately five times the mean current pulse size difference for racemic (x = 0) and L-only (x = +1) cystine samples. A similar result was observed for aspartate. Our discovery opens up new opportunities for micro/nanoscopic chiral amino acid analysis, which can potentially be used in single-cell analysis.


Subject(s)
Amino Acids , Crystallization , Stereoisomerism , Amino Acids/chemistry , Cystine/chemistry , Cadmium/chemistry , Aspartic Acid/chemistry , Metals/chemistry
4.
J Agric Food Chem ; 72(17): 9937-9946, 2024 May 01.
Article in English | MEDLINE | ID: mdl-38651303

ABSTRACT

The engineered human cystathionine-γ-lyase (hCGL) resulting in enhanced activity toward both cysteine and cystine unveils a potential robust antitumor activity. However, the presence of cysteine residues has the potential to induce oligomerization or incorrect disulfide bonding, which may decrease the bioavailability of biopharmaceuticals. Through a meticulous design process targeting the cysteine residues within engineered hCGL, a set of potential beneficial mutants were obtained by virtual screening employing Rosetta and ABACUS. Experimental measurements have revealed that most of the mutants showed increased activity toward both substrates l-Cys and CSSC. Furthermore, mutants C109V and C229D demonstrated Tm value increases of 8.2 and 1.8 °C, respectively. After an 80 min incubation at 60 °C, mutant C229D still maintained high residual activity. Unexpectedly, mutant C109V, displaying activity approximately 2-fold higher than the activity of wild type (WT) for both substrates, showed disappointing instability in plasma, which suggests that computational design still requires further consideration. Analysis of their structure and molecular dynamics (MD) simulation revealed the impact of hydrophobic interaction, hydrogen bonds, and near-attack conformation (NAC) stability on activity and stability. This study acquired information about mutants that exhibit enhanced activity or thermal resistance and serve as valuable guidance for subsequent specific cysteine modifications.


Subject(s)
Cystathionine gamma-Lyase , Cysteine , Molecular Dynamics Simulation , Protein Engineering , Cysteine/chemistry , Cysteine/metabolism , Humans , Cystathionine gamma-Lyase/genetics , Cystathionine gamma-Lyase/chemistry , Cystathionine gamma-Lyase/metabolism , Enzyme Stability , Cystine/chemistry , Hydrogen Bonding , Mutation , Kinetics
5.
J Phys Chem B ; 128(13): 3145-3156, 2024 Apr 04.
Article in English | MEDLINE | ID: mdl-38512062

ABSTRACT

In this study, a three-layered multicenter ONIOM approach is implemented to characterize the naive folding pathway of bovine pancreatic trypsin inhibitor (BPTI). Each layer represents a distinct level of theory, where the initial layer, encompassing the entire protein, is modeled by a general all-atom force-field GFN-FF. An intermediate electronic structure layer consisting of three multicenter fragments is introduced with the state-of-the-art semiempirical tight-binding method GFN2-xTB. Higher accuracy, specifically addressing the breaking and formation of the three disulfide bonds, is achieved at the innermost layer using the composite DFT method r2SCAN-3c. Our analysis sheds light on the structural stability of BPTI, particularly the significance of interlinking disulfide bonds. The accuracy and efficiency of the multicenter QM/SQM/MM approach are benchmarked using the oxidative formation of cystine. For the folding pathway of BPTI, relative stabilities are investigated through the calculation of free energy contributions for selected intermediates, focusing on the impact of the disulfide bond. Our results highlight the intricate trade-off between accuracy and computational cost, demonstrating that the multicenter ONIOM approach provides a well-balanced and comprehensive solution to describe electronic structure effects in biomolecular systems. We conclude that multiscale energy landscape exploration provides a robust methodology for the study of intriguing biological targets.


Subject(s)
Disulfides , Protein Folding , Animals , Cattle , Aprotinin/chemistry , Cystine/chemistry , Disulfides/chemistry , Proteins
6.
J Mol Graph Model ; 125: 108607, 2023 12.
Article in English | MEDLINE | ID: mdl-37634277

ABSTRACT

The current study involves the investigation of reducing properties of disulfide bonded heteraphanes. The calculated adiabatic electron affinity (AEA) values of heteraphanes are found comparable to that of cystine molecule and are capable of undergoing reversible redox reactions. In aqueous phase, these show high propensity to get reduced. The reaction energies calculated using isodesmic equations reflect the strain associated with the studied thiacyclophane models. Increase in the number of disulfide bonds results in less strain and more stabilization. Through-space transannular interactions in the selected heteraphanes have a decisive influence on the structure stabilization associated with the systems. The results reported in the current study are expected to play a vital role while designing redox driven drug carriers by incorporating these systems in biomolecules.


Subject(s)
Cystine , Disulfides , Models, Molecular , Cystine/chemistry , Disulfides/chemistry , Oxidation-Reduction
7.
ACS Appl Mater Interfaces ; 15(27): 32177-32187, 2023 Jul 12.
Article in English | MEDLINE | ID: mdl-37387421

ABSTRACT

The self-association of metabolites into well-ordered assemblies at the nanoscale has significant biological and medical implications. The thiol-containing amino acid cysteine (CYS) can assemble into amyloid-like nanofibrils, and its oxidized form, the disulfide-bonded cystine (CTE), forms hexagonal crystals as those found in cystinuria due to metabolic disorder. Yet, there have been no attempts to connect these two phenomena, especially the fibril-to-crystal transition. Here, we reveal that these are not separated events, and the CYS-forming amyloid fibrils are mechanistically linked to hexagonal CTE crystals. For the first time, we demonstrated that cysteine fibrils are a prerequisite for forming cystine crystals, as observed experimentally. To further understand this mechanism, we studied the effects of thiol-containing cystinuria drugs (tiopronin, TIO; and d-penicillamine, PEN) and the canonical epigallocatechin gallate (EGCG) amyloid inhibitor on fibril formation by CYS. The thiol-containing drugs do not solely interact with monomeric CYS via disulfide bond formation but can disrupt amyloid formation by targeting CYS oligomers. On the other hand, EGCG forms inhibitor-dominant complexes (more than one EGCG molecule per cysteine unit) to prevent CYS fibril formation. Interestingly, while CYS can be oxidized into CTE, the thiol drugs can reduce CTE back to CYS. We thus suggest that the formation of crystals in cystinuria could be halted at the initial stage by targeting CYS fibril formation as an alternative to solubilizing the water-insoluble hexagonal CTE crystals at a later stage. Taken together, we depicted a complex hierarchical organization in a simple amino acid assembly with implications for therapeutic intervention.


Subject(s)
Cysteine , Cystinuria , Humans , Cysteine/chemistry , Cystine/chemistry , Cystinuria/drug therapy , Amino Acids/therapeutic use , Amyloid/chemistry , Disulfides/therapeutic use
8.
Chem Commun (Camb) ; 59(45): 6917-6920, 2023 Jun 01.
Article in English | MEDLINE | ID: mdl-37200079

ABSTRACT

Ru-Alkylidene catalysed olefin metathesis generates metabolically stable cystine bridge peptidomimetics with defined geometry. Deleterious coordinative bonding to the catalyst by sulfur-containing functionality found in cysteine and methionine residues can be negated by in situ and reversible oxidation of thiol and thioether functionality, as disulfides and S-oxides respectively, to facilitate high yielding ring-closing and cross metathesis of bioorthogonally protected peptides.


Subject(s)
Cysteine , Methionine , Cysteine/chemistry , Methionine/chemistry , Peptides/chemistry , Cystine/chemistry , Racemethionine
9.
Proteins ; 91(2): 256-267, 2023 02.
Article in English | MEDLINE | ID: mdl-36107799

ABSTRACT

The archetypal Viola odorata cyclotide cycloviolacin-O1 and its seven analogs, created by partial or total reduction of the three native S-S linkages belonging to the "cyclic cystine knot" (CCK) motif are studied for their structural and dynamical diversities using molecular dynamics simulations. The results indicate interesting interplay between the constraints imposed by the S-S bonds on the dynamical modes and the corresponding structure of the model peptide. Principal component analysis brings out the variation in the extent of dynamical freedom along the peptide backbone for each model. The motions are characterized by low amplitude diffusive modes in the peptides retaining most of the native S-S linkages in contrast to the large amplitude discrete jumps where at least two or all of the three S-S linkages are reduced. Simulation results further indicate that the disulfide bond between Cys1-18 is formed at a much faster pace compared with its two other peers Cys5-20 and Cys10-25 as found in the native peptide. This gives insight as to why the S-S linkages appear in the native peptide in a particular combination. Model therapeutics and drug delivery engines can potentially utilize this information to customize the engineered S-S bonds and gauge its impact on the dynamic flexibility of a model macrocyclic peptide.


Subject(s)
Cyclotides , Cyclotides/chemistry , Cystine/chemistry , Amino Acid Sequence , Models, Molecular
10.
Br J Ophthalmol ; 107(2): 234-241, 2023 Feb.
Article in English | MEDLINE | ID: mdl-34531199

ABSTRACT

PRÉCIS: Cystinosis is a lysosomal storage disease leading to an accumulation of cystine crystals in several organs. We aim to comprehensively describe chorioretinal cystine crystals via spectral domain optical coherence tomography (SD-OCT) and elaborate a new biomarker for systemic disease control. BACKGROUND/AIMS: Cystinosis is a rare lysosomal storage disease leading to an accumulation of cystine crystals in several organs. This study aims to describe the deposition of retinochoroidal crystals in infantile nephropathic cystinosis and to elucidate their potential value as an objective biomarker for systemic disease control. METHODS: This cross-sectional study was carried out by the University Eye Hospital of the Ludwig-Maximilian University (Munich, Germany) in collaboration with the German Cystinosis Study Group. A complete ophthalmologic examination was performed, along with posterior segment SD-OCT (Spectralis; Heidelberg Engineering GmbH, Heidelberg, Germany). Retinochoroidal crystals were graded by employing a novel semiquantitative grading system-the retinochoroidal cystine crystal score (RCCCS). To quantify quality of vision, patients completed a specific questionnaire. A total of 85 eyes of 43 patients with cystinosis were included (mean age 22.3±8.8 years, range 6-39; male:female ratio=23:20). RESULTS: Cystine crystals were detectable in all neuroretinal layers and the choroid, most frequently in the choriocapillaris. The RCCCS was negatively correlated with cysteamine intake (r=0.533, p=0.001) and positively with cystatin C, a stable parameter of renal function (r=0.496, p=0.016). Moreover, the value of the RCCCS affected subjective quality of vision. Genetic analysis indicated pronounced crystal deposition in patients with heterozygous mutations containing the 57-kb-deletion allele of the CTNS gene. CONCLUSION: Ocular cystinosis leads to retinochoroidal crystal accumulation in every stage of the disease. Crystal deposition may be markedly influenced by oral cysteamine therapy. Therefore, the presented SD-OCT based grading system might serve as an objective biomarker for systemic disease control.


Subject(s)
Cystinosis , Humans , Male , Female , Child , Adolescent , Young Adult , Adult , Cystinosis/diagnosis , Cysteamine , Cystine/chemistry , Tomography, Optical Coherence/methods , Cross-Sectional Studies , Cornea
11.
Toxicon ; 219: 106926, 2022 Nov.
Article in English | MEDLINE | ID: mdl-36167143

ABSTRACT

The inhibitory cystine knot (ICK) motif is an evolutionarily optimized disulfide-rich peptide motif widely present in diverse phyla with distinct biological functions. Cysteine disulfides are highly conserved in the ICK motif with C1-C4 (Disulfide-I), C2-C5(Disulfide-II), and C3-C6(Disulfide-III) connectivities in a sequence. Disulfide-I and disulfide-II form a loop and the disulfide-III tethers through the loop forming a knotted fold. The current report has analysed the conformation of disulfides in the ICK motif using the side-chain torsional angles of cysteine disulfide. In crystal structures: 88% of Disulfide-I have (+,-)SynRHHook, 92% of Disulfide-II have (+,-)RHSpiral, and 100% of Disulfide-III have (-,-)LHSpiral conformations. In NMR structures, conformational diversity has been observed for each of the cysteine disulfides of the ICK motif. The highest percentage occurrence in NMR structures: 27% of Disulfide-I have (+,-)SynRHHook, 36% of Disulfide-II have (+,-)RHSpiral, and 50% of Disulfide-III have (-,-)LHSpiral conformations. In the view of the method of identification of disulfides between cysteine residues using NMR spectroscopy, the NMR structure represents an ensemble of conformations of disulfides instead of specific disulfide conformation. The retention of the conformation in both X-ray and NMR structures supports the conservation of conformation of disulfides in the ICK motif. The tendency to exhibit specific conformation of disulfide even with variations in 3D structures supports the evolutionarily optimized nature of the ICK motif.


Subject(s)
Cystine , Disulfides , Disulfides/chemistry , Cystine/chemistry , Cysteine/chemistry , Protein Conformation , Peptides/chemistry
12.
Orv Hetil ; 163(21): 846-852, 2022 May 22.
Article in Hungarian | MEDLINE | ID: mdl-35598215

ABSTRACT

Cystinosis is a rare lysosomal storage disease affecting amino acid metabolism, characterized by the accumulation and crystallization of cystine in various tissues, primarily in the eye and kidney. The major ophthalmic symptom is photophobia, which is related to the corneal deposition of cystine crystals. The light sensitivity significantly impairs the quality of life of the affected patients, thus, effective ophthalmic treatment to reduce the crystal density is very importance. In the current case report, we present the characteristic ocular clinical appearance and treatment options of cystinosis by reviewing the literature. A simple aqueous solution of cysteamine, which aids in the dissolution of crystals, has been widely used in topical treatment in the past, however, its therapeutic efficacy is debatable. Recently, a new viscous formulation of cysteamine has been proposed for ophthalmic treatment. For the treatment of corneal cystine crystals in our patient, the new viscous format of cysteamine has been applied, and therapeutic effects were recorded for a year. Applying the viscous cysteamine formulation, a marked and gradual decrease in photophobia was observed in our patient in the first year of the treatment. Anterior-segment optical coherence tomography and in vivo confocal microscopy represented a continuous decrease in the density of corneal crystals even from the first month of the treatment period. The aim of our case report is to present the ophthalmic symptoms of cystinosis and the results of the first clinical application of viscous formulation of cysteamin eye drops in Hungary in a cystinosis patient.


Subject(s)
Corneal Diseases , Cystinosis , Cornea/metabolism , Corneal Diseases/diagnosis , Corneal Diseases/drug therapy , Corneal Diseases/etiology , Cysteamine/metabolism , Cysteamine/therapeutic use , Cystine/chemistry , Cystine/metabolism , Cystine/therapeutic use , Cystinosis/complications , Cystinosis/diagnosis , Cystinosis/drug therapy , Humans , Photophobia/etiology , Quality of Life , Visual Acuity
13.
Amino Acids ; 54(8): 1123-1133, 2022 Aug.
Article in English | MEDLINE | ID: mdl-35296914

ABSTRACT

It is assumed that genetic diseases affecting the metabolism of cysteine and the kidney function lead to two different kinds of pathologies, namely cystinuria and cystinosis whereby generate L-cystine crystals. Recently, the presence of L-cysteine crystal has been underlined in the case of cystinosis. Interestingly, it can be strikingly seen that cystine ([-S-CH2-CH-(NH2)-COOH]2) consists of two cysteine (C3H7NO2S) molecules connected by a disulfide (S-S) bond. Therefore, the study of cystine and cysteine is important for providing a better understanding of cystinuria and cystinosis. In this paper, we elucidate the discrepancy between L-cystine and L-cysteine by investigating the theoretical and experimental infrared spectra (IR), X-ray diffraction (XRD) as well as Raman spectra aiming to obtain a better characterization of abnormal deposits related to these two genetic pathologies.


Subject(s)
Cystinosis , Cystinuria , Cysteine/chemistry , Cystine/chemistry , Disulfides , Humans
14.
Molecules ; 27(4)2022 Feb 15.
Article in English | MEDLINE | ID: mdl-35209090

ABSTRACT

Chemo and siRNA synergic treatments for tumors is a promising new therapeutic trend. Selenocystine, a selenium analog of cysteine, has been considered a potential antitumor agent due to its redox perturbing role. In this study, we developed a nanocarrier for siRNA based on a selenocystine analog engineered polyetherimide and achieved traceable siRNA delivery and the synergic killing of tumor cells. Notably, we applied the label-free Schiff base fluorescence mechanism, which enabled us to trace the siRNA delivery and to monitor the selenocystine analogs' local performance. A novel selenocystine-derived fluorescent Schiff base linker was used to crosslink the polyetherimide, thereby generating a traceable siRNA delivery vehicle with green fluorescence. Moreover, we found that this compound induced tumor cells to undergo senescence. Together with the delivery of a siRNA targeting the anti-apoptotic BCL-xl/w genes in senescent cells, it achieved a synergistic inhibition function by inducing both senescence and apoptosis of tumor cells. Therefore, this study provides insights into the development of label-free probes, prodrugs, and materials towards the synergic strategies for cancer therapy.


Subject(s)
Cystine/analogs & derivatives , Drug Delivery Systems , Gene Transfer Techniques , Nanocomposites/chemistry , Organoselenium Compounds/chemistry , RNA, Small Interfering/genetics , Schiff Bases/chemistry , Cell Line, Tumor , Cell Survival , Cystine/chemistry , Fluorescence , Humans , Microscopy, Fluorescence , Molecular Structure , RNA, Small Interfering/administration & dosage
15.
J Phys Chem A ; 126(9): 1496-1503, 2022 Mar 10.
Article in English | MEDLINE | ID: mdl-35213156

ABSTRACT

We demonstrate site-specific X-ray induced fragmentation across the sulfur L-edge of protonated cystine, the dimer of the amino acid cysteine. Ion yield NEXAFS were performed in the gas phase using electrospray ionization (ESI) in combination with an ion trap. The interpretation of the sulfur L-edge NEXAFS spectrum is supported by Restricted Open-Shell Configuration Interaction (ROCIS) calculations. The fragmentation pathway of triply charged cystine ions was modeled by Molecular Dynamics (MD) simulations. We have deduced a possible pathway of fragmentation upon excitation and ionization of S 2p electrons. The disulfide bridge breaks for resonant excitation at lower photon energies but remains intact upon higher energy resonant excitation and upon ionization of S 2p. The larger fragments initially formed subsequently break into smaller fragments.


Subject(s)
Cysteine , Cystine , Cysteine/chemistry , Cystine/chemistry , Electrons , Ions , Spectrometry, Mass, Electrospray Ionization , X-Rays
16.
Acc Chem Res ; 55(4): 516-525, 2022 02 15.
Article in English | MEDLINE | ID: mdl-35088591

ABSTRACT

Aberrant crystallization within the human body can lead to several disease states or adverse outcomes, yet much remains to be understood about the critical stages leading to these events, which can include crystal nucleation and growth, crystal aggregation, and the adhesion of crystals to cells. Kidney stones, which are aggregates of single crystals with physiological origins, are particularly illustrative of pathological crystallization, with 10% of the U.S. population experiencing at least one stone occurrence in their lifetimes. The human record of kidney stones is more than 2000 years old, as noted by Hippocrates in his renowned oath and much later by Robert Hooke in his treatise Micrographia. William Hyde Wollaston, who was a physician, chemist, physicist, and crystallographer, was fascinated with stones, leading him to discover an unusual stone that he described in 1810 as cystic oxide, later corrected to cystine. Despite this long history, however, a fundamental understanding of the stages of stone formation and the rational design of therapies for stone prevention have remained elusive.This Account reviews discoveries and advances from our laboratories that have unraveled the complex crystal growth mechanisms of l-cystine, which forms l-cystine kidney stones in at least 20 000 individuals in the U.S. alone. Although l-cystine stones affect fewer individuals than common calcium oxalate stones, they are usually larger, recur more frequently, and are more likely to cause chronic kidney disease. Real-time in situ atomic force microscopy (AFM) reveals that the crystal growth of hexagonal l-cystine is characterized by a complex mechanism in which six interlaced anisotropic spirals grow synchronously, emanating from a single screw dislocation to generate a micromorphology with the appearance of stacked hexagonal islands. In contrast, proximal heterochiral dislocations produce features that appear to be spirals but actually are closed loops, akin to a Frank-Read source. These unusual and aesthetic growth patterns can be explained by the coincidence of the dislocation Burgers vector and the crystallographic 61 screw axis. Inhibiting l-cystine crystal growth is key to preventing stone formation. Decades of studies of "tailor-made additives", which are imposter molecules that closely resemble the solute and bind to crystal faces through molecular recognition, have demonstrated their effects on crystal properties such as morphology and polymorphism. The ability to visualize crystal growth in real time by AFM enables quantitative measurements of step velocities and, by extension, the effect of prospective inhibitors on growth rates, which can then be used to deduce inhibition mechanisms. Investigations with a wide range of prospective inhibitors revealed the importance of precise molecular recognition for binding l-cystine imposters to crystal sites, which results in step pinning and the inhibition of step advancement as well as the growth of bulk crystals. Moreover, select inhibitors of crystal growth, measured in vitro, reduce or eliminate stone formation in knockout mouse models of cystinuria, promising a new pathway to l-cystine stone prevention. These observations have wide-ranging implications for the design of therapies based on tailor-made additives for diseases associated with aberrant crystallization, from disease-related stones to "xenostones" that form in vivo because of the crystallization of low-solubility therapeutic agents such as antiretroviral agents.


Subject(s)
Cystinuria , Kidney Calculi , Animals , Crystallization , Cystine/chemistry , Cystine/metabolism , Cystine/therapeutic use , Cystinuria/complications , Cystinuria/drug therapy , Cystinuria/metabolism , Kidney , Kidney Calculi/chemistry , Kidney Calculi/etiology , Kidney Calculi/prevention & control , Male , Mice
17.
J Med Chem ; 65(1): 485-496, 2022 01 13.
Article in English | MEDLINE | ID: mdl-34931831

ABSTRACT

Inhibitor cystine knot peptides, derived from venom, have evolved to block ion channel function but are often toxic when dosed at pharmacologically relevant levels in vivo. The article describes the design of analogues of ProTx-II that safely display systemic in vivo blocking of Nav1.7, resulting in a latency of response to thermal stimuli in rodents. The new designs achieve a better in vivo profile by improving ion channel selectivity and limiting the ability of the peptides to cause mast cell degranulation. The design rationale, structural modeling, in vitro profiles, and rat tail flick outcomes are disclosed and discussed.


Subject(s)
NAV1.7 Voltage-Gated Sodium Channel/drug effects , Pain/drug therapy , Sodium Channel Blockers/chemical synthesis , Sodium Channel Blockers/pharmacology , Spider Venoms/chemical synthesis , Animals , Cell Degranulation/drug effects , Cystine/chemistry , Drug Design , Hot Temperature , Mast Cells/drug effects , Models, Molecular , Pain Measurement/drug effects , Rats , Spider Venoms/pharmacology
18.
Nat Commun ; 12(1): 5672, 2021 09 28.
Article in English | MEDLINE | ID: mdl-34584078

ABSTRACT

Nature forms S-S bonds by oxidizing two sulfhydryl groups, and no enzyme installing an intact hydropersulfide (-SSH) group into a natural product has been identified to date. The leinamycin (LNM) family of natural products features intact S-S bonds, and previously we reported an SH domain (LnmJ-SH) within the LNM hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line as a cysteine lyase that plays a role in sulfur incorporation. Here we report the characterization of an S-adenosyl methionine (SAM)-dependent hydropersulfide methyltransferase (GnmP) for guangnanmycin (GNM) biosynthesis, discovery of hydropersulfides as the nascent products of the GNM and LNM hybrid NRPS-PKS assembly lines, and revelation of three SH domains (GnmT-SH, LnmJ-SH, and WsmR-SH) within the GNM, LNM, and weishanmycin (WSM) hybrid NRPS-PKS assembly lines as thiocysteine lyases. Based on these findings, we propose a biosynthetic model for the LNM family of natural products, featuring thiocysteine lyases as PKS domains that directly install a -SSH group into the GNM, LNM, or WSM polyketide scaffold. Genome mining reveals that SH domains are widespread in Nature, extending beyond the LNM family of natural products. The SH domains could also be leveraged as biocatalysts to install an -SSH group into other biologically relevant scaffolds.


Subject(s)
Biological Products/metabolism , Carbon-Sulfur Lyases/metabolism , Cysteine/analogs & derivatives , Methyltransferases/metabolism , Polyketide Synthases/metabolism , Sulfides/metabolism , Animals , Biological Products/chemistry , Cysteine/metabolism , Cystine/chemistry , Cystine/metabolism , Humans , Lactams/chemical synthesis , Lactams/chemistry , Lactams/metabolism , Macrolides/chemical synthesis , Macrolides/chemistry , Macrolides/metabolism , Models, Chemical , Molecular Structure , Peptide Synthases/metabolism , Streptomyces/genetics , Streptomyces/metabolism , Substrate Specificity , Sulfides/chemistry , Thiazoles/chemical synthesis , Thiazoles/chemistry , Thiazoles/metabolism , Thiones/chemical synthesis , Thiones/chemistry , Thiones/metabolism , src Homology Domains
19.
Toxins (Basel) ; 13(9)2021 09 03.
Article in English | MEDLINE | ID: mdl-34564625

ABSTRACT

Inhibitor cystine knot (ICK) peptides are knotted peptides with three intramolecular disulfide bonds that affect several types of ion channels. Some are proteolytically stable and are promising scaffolds for drug development. GTx1-15 is an ICK peptide that inhibits the voltage-dependent calcium channel Cav3.1 and the voltage-dependent sodium channels Nav1.3 and Nav1.7. As a model molecule to develop an ICK peptide drug, we investigated several important pharmaceutical characteristics of GTx1-15. The stability of GTx1-15 in rat and human blood plasma was examined, and no GTx1-15 degradation was observed in either rat or human blood plasma for 24 h in vitro. GTx1-15 in blood circulation was detected for several hours after intravenous and intramuscular administration, indicating high stability in plasma. The thermal stability of GTx1-15 as examined by high thermal incubation and protein thermal shift assays indicated that GTx1-15 possesses high heat stability. The cytotoxicity and immunogenicity of GTx1-15 were examined using the human monocytic leukemia cell line THP-1. GTx1-15 showed no cytotoxicity or immunogenicity even at high concentrations. These results indicate that GTx1-15 itself is suitable for peptide drug development and as a peptide library scaffold.


Subject(s)
Cystine/chemistry , Peptides/chemistry , Plasma/drug effects , Spider Venoms/adverse effects , Spiders/chemistry , Animals , Humans , Injections, Intramuscular , Injections, Intravenous , Rats , THP-1 Cells
20.
Molecules ; 26(17)2021 Aug 27.
Article in English | MEDLINE | ID: mdl-34500638

ABSTRACT

The effect of humidity on sheep wool during irradiation by an accelerated electron beam was examined. Each of the samples with 10%, 53%, and 97% relative humidity (RH) absorbed a dose of 0, 109, and 257 kGy, respectively. After being freely kept in common laboratory conditions, the samples were subjected to batch Co(II) sorption experiments monitored with VIS spectrometry for different lapses from electron beam exposure. Along with the sorption, FTIR spectral analysis of the wool samples was conducted for cysteic acid and cystine monoxide, and later, the examination was completed, with pH measuring 0.05 molar KCl extract from the wool samples. Besides a relationship to the absorbed dose and lapse, the sorptivity results showed considerable dependence on wool humidity under exposure. When humidity was deficient (10% RH), the sorptivity was lower due to limited transformation of cystine monoxide to cysteic acid. The wool pre-conditioned at 53% RH, which is the humidity close to common environmental conditions, demonstrated the best Co(II) sorptivity in any case. This finding enables the elimination of pre-exposure wool conditioning in practice. Under excessive humidity of 97% RH and enough high dose of 257 kGy, radiolysis of water occurred, deteriorating the sorptivity. Each wool humidity, dose, and lapse showed a particular scenario. The time and humidity variations in the sorptivity for the non-irradiated sample were a little surprising; despite the absence of electron irradiation, relevant results indicated a strong sensitivity to pre-condition humidity and lapse from the start of the monitoring.


Subject(s)
Cobalt/chemistry , Ions/chemistry , Sheep/metabolism , Wool/chemistry , Adsorption/physiology , Animals , Cystine/chemistry , Electrons , Humidity , Water/chemistry
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