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1.
J Am Chem Soc ; 2024 Jul 12.
Article in English | MEDLINE | ID: mdl-38994607

ABSTRACT

Interests in covalent drugs have grown in modern drug discovery as they could tackle challenging targets traditionally considered "undruggable". The identification of covalent binders to target proteins typically involves directly measuring protein covalent modifications using high-resolution mass spectrometry. With a continually expanding library of compounds, conventional mass spectrometry platforms such as LC-MS and SPE-MS have become limiting factors for high-throughput screening. Here, we introduce a prototype high-resolution acoustic ejection mass spectrometry (AEMS) system for the rapid screening of a covalent modifier library comprising ∼10,000 compounds against a 50 kDa-sized target protein─Werner syndrome helicase. The screening samples were arranged in a 1536-well format. The sample buffer containing high-concentration salts was directly analyzed without any cleanup steps, minimizing sample preparation efforts and ensuring protein stability. The entire AEMS analysis process could be completed within a mere 17 h. An automated data analysis tool facilitated batch processing of the sample data and quantitation of the formation of various covalent protein-ligand adducts. The screening results displayed a high degree of fidelity, with a Z' factor of 0.8 and a hit rate of 2.3%. The identified hits underwent orthogonal testing in a biochemical activity assay, revealing that 75% were functional antagonists of the target protein. Notably, a comparative analysis with LC-MS showcased the AEMS platform's low risk of false positives or false negatives. This innovative platform has enabled robust high-throughput covalent modifier screening, featuring a 10-fold increase in library size and a 10- to 100-fold increase in throughput when compared with similar reports in the existing literature.

2.
PLoS One ; 7(11): e49757, 2012.
Article in English | MEDLINE | ID: mdl-23152933

ABSTRACT

We explored the potential of mutant allele-specific gene silencing (ASGS) in providing therapeutic benefit in two established mouse models of the autosomal dominantly-inherited muscle disorders, Malignant Hyperthermia (MH) and Central Core Disease (CCD). Candidate ASGS siRNAs were designed and validated for efficacy and specificity on ryanodine receptor (RyR1) cDNA mini-constructs expressed in HEK293 cells using RT-PCR- and confocal microscopy-based assays. In vivo delivery of the most efficacious identified siRNAs into flexor digitorum brevis (FDB) muscles was achieved by injection/electroporation of footpads of 4-6 month old heterozygous Ryr1(Y524S/+) (YS/+) and Ryr1(I4895T/+) (IT/+) knock-in mice, established mouse models of MH with cores and CCD, respectively. Treatment of IT/+ mice resulted in a modest rescue of deficits in the maximum rate (∼38% rescue) and magnitude (∼78%) of ligand-induced Ca(2+) release that occurred in the absence of a change in the magnitude of electrically-evoked Ca(2+) release. Compared to the difference between the caffeine sensitivity of Ca(2+) release in FDB fibers from YS/+ and WT mice treated with SCR siRNA (EC(50): 1.1 mM versus 4.4 mM, respectively), caffeine sensitivity was normalized in FDB fibers from YS/+ mice following 2 (EC(50): 2.8 mM) and 4 week (EC(50): 6.6 mM) treatment with YS allele-specific siRNA. Moreover, the temperature-dependent increase in resting Ca(2+) observed in FDB fibers from YS/+ mice was normalized to WT levels after 2 weeks of treatment with YS allele-specific siRNA. As determined by quantitative real time PCR, the degree of functional rescue in YS/+ and IT/+ mice correlated well with the relative increase in fractional WT allele expression.


Subject(s)
Alleles , Gene Silencing , Genes, Dominant/genetics , Muscles/pathology , Muscular Diseases/genetics , Muscular Diseases/pathology , Animals , Disease Models, Animal , Gene Knock-In Techniques , Gene Knockdown Techniques , Genetic Testing , HEK293 Cells , Humans , Mice , Mutation/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Ryanodine Receptor Calcium Release Channel/metabolism
3.
J Gen Physiol ; 137(1): 43-57, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21149547

ABSTRACT

The type 1 isoform of the ryanodine receptor (RYR1) is the Ca(2+) release channel of the sarcoplasmic reticulum (SR) that is activated during skeletal muscle excitation-contraction (EC) coupling. Mutations in the RYR1 gene cause several rare inherited skeletal muscle disorders, including malignant hyperthermia and central core disease (CCD). The human RYR1(I4898T) mutation is one of the most common CCD mutations. To elucidate the mechanism by which RYR1 function is altered by this mutation, we characterized in vivo muscle strength, EC coupling, SR Ca(2+) content, and RYR1 Ca(2+) release channel function using adult heterozygous Ryr1(I4895T/+) knock-in mice (IT/+). Compared with age-matched wild-type (WT) mice, IT/+ mice exhibited significantly reduced upper body and grip strength. In spite of normal total SR Ca(2+) content, both electrically evoked and 4-chloro-m-cresol-induced Ca(2+) release were significantly reduced and slowed in single intact flexor digitorum brevis fibers isolated from 4-6-mo-old IT/+ mice. The sensitivity of the SR Ca(2+) release mechanism to activation was not enhanced in fibers of IT/+ mice. Single-channel measurements of purified recombinant channels incorporated in planar lipid bilayers revealed that Ca(2+) permeation was abolished for homotetrameric IT channels and significantly reduced for heterotetrameric WT:IT channels. Collectively, these findings indicate that in vivo muscle weakness observed in IT/+ knock-in mice arises from a reduction in the magnitude and rate of RYR1 Ca(2+) release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca(2+) ion permeation.


Subject(s)
Calcium/metabolism , Muscle Weakness/genetics , Muscle Weakness/metabolism , Ryanodine Receptor Calcium Release Channel/genetics , Sarcoplasmic Reticulum/metabolism , Animals , Calcium Channels/metabolism , Calcium Signaling/genetics , Cresols/metabolism , Gene Knock-In Techniques , HEK293 Cells , Humans , Male , Mice , Mice, Inbred Strains , Muscle Contraction/genetics , Muscle Contraction/physiology , Muscle Strength/genetics , Muscle Strength/physiology , Muscle, Skeletal/metabolism , Muscle, Skeletal/physiopathology , Mutation , Ryanodine Receptor Calcium Release Channel/deficiency , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic Reticulum/genetics
4.
Aging Cell ; 9(6): 958-70, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20961389

ABSTRACT

The I4898T (IT) mutation in type 1 ryanodine receptor (RyR1), the Ca(2+) release channel of the sarcoplasmic reticulum (SR) is linked to a form of central core disease (CCD) in humans and results in a nonleaky channel and excitation-contraction uncoupling. We characterized age-dependent and fiber-type-dependent alterations in muscle ultrastructure, as well as the magnitude and spatiotemporal properties of evoked Ca(2+) release in heterozygous Ryr1(I4895T/WT) (IT/+) knock-in mice on a mixed genetic background. The results indicate a classical but mild CCD phenotype that includes muscle weakness and the presence of mitochondrial-deficient areas in type I fibers. Electrically evoked Ca(2+) release is significantly reduced in single flexor digitorum brevis (FDB) fibers from young and old IT/+ mice. Structural changes are strongly fiber-type specific, affecting type I and IIB/IIX fibers in very distinct ways, and sparing type IIA fibers. Ultrastructural alterations in our IT/+ mice are also present in wild type, but at a lower frequency and older ages, suggesting that the disease mutation on the mixed background promotes an acceleration of normal age-dependent changes. The observed functional and structural alterations and their similarity to age-associated changes are entirely consistent with the known properties of the mutated channel, which result in reduced calcium release as is also observed in normal aging muscle. In strong contrast to these observations, a subset of patients with the analogous human heterozygous mutation and IT/+ mice on an inbred 129S2/SvPasCrl background exhibit a more severe disease phenotype, which is not directly consistent with the mutated channel properties.


Subject(s)
Aging, Premature/genetics , Muscle Fibers, Skeletal/physiology , Mutation , Ryanodine Receptor Calcium Release Channel/genetics , Animals , Calcium/metabolism , Electrophysiological Phenomena , Mice , Mice, Knockout , Ryanodine Receptor Calcium Release Channel/metabolism
5.
Neuromuscul Disord ; 20(3): 166-73, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20080402

ABSTRACT

The skeletal muscle ryanodine receptor plays a crucial role in excitation-contraction (EC) coupling and is implicated in various congenital myopathies. The periodic paralyses are a heterogeneous, dominantly inherited group of conditions mainly associated with mutations in the SCN4A and the CACNA1S genes. The interaction between RyR1 and DHPR proteins underlies depolarization-induced Ca(2+) release during EC coupling in skeletal muscle. We report a 35-year-old woman presenting with signs and symptoms of a congenital myopathy at birth and repeated episodes of generalized, atypical normokalaemic paralysis in her late teens. Genetic studies of this patient revealed three heterozygous RYR1 substitutions (p.Arg2241X, p.Asp708Asn and p.Arg2939Lys) associated with marked reduction of the RyR1 protein and abnormal DHPR distribution. We conclude that RYR1 mutations may give rise to both myopathies and atypical periodic paralysis, and RYR1 mutations may underlie other unresolved cases of periodic paralysis with unusual features.


Subject(s)
Muscle, Skeletal/pathology , Muscular Diseases/genetics , Muscular Diseases/pathology , Mutation/genetics , Ryanodine Receptor Calcium Release Channel/genetics , Adult , Arginine/genetics , Caffeine/pharmacology , Calcium/metabolism , Calcium Channels/genetics , Calcium Channels, L-Type/genetics , Calcium Channels, L-Type/metabolism , Cell Line, Transformed , DNA Mutational Analysis/methods , Electron Transport Complex IV/drug effects , Family Health , Female , Humans , Lysine/genetics , Male , Membrane Potentials/drug effects , Membrane Potentials/genetics , Muscular Diseases/classification , NAV1.4 Voltage-Gated Sodium Channel , Patch-Clamp Techniques , Phosphodiesterase Inhibitors/pharmacology , Ryanodine/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Sodium Channels/genetics , Transfection/methods , Tritium/metabolism
6.
Proc Natl Acad Sci U S A ; 106(13): 5135-40, 2009 Mar 31.
Article in English | MEDLINE | ID: mdl-19279214

ABSTRACT

Voltage-dependent calcium channels (Ca(V)) open in response to changes in membrane potential, but their activity is modulated by Ca(2+) binding to calmodulin (CaM). Structural studies of this family of channels have focused on CaM bound to the IQ motif; however, the minimal differences between structures cannot adequately describe CaM's role in the regulation of these channels. We report a unique crystal structure of a 77-residue fragment of the Ca(V)1.2 alpha(1) subunit carboxyl terminus, which includes a tandem of the pre-IQ and IQ domains, in complex with Ca(2+).CaM in 2 distinct binding modes. The structure of the Ca(V)1.2 fragment is an unusual dimer of 2 coiled-coiled pre-IQ regions bridged by 2 Ca(2+).CaMs interacting with the pre-IQ regions and a canonical Ca(V)1-IQ-Ca(2+).CaM complex. Native Ca(V)1.2 channels are shown to be a mixture of monomers/dimers and a point mutation in the pre-IQ region predicted to abolish the coiled-coil structure significantly reduces Ca(2+)-dependent inactivation of heterologously expressed Ca(V)1.2 channels.


Subject(s)
Calcium Channels, L-Type/chemistry , Calmodulin/chemistry , Calcium-Binding Proteins/chemistry , Crystallography, X-Ray , Dimerization , Humans , Point Mutation , Protein Structure, Tertiary
7.
Proc Natl Acad Sci U S A ; 104(47): 18537-42, 2007 Nov 20.
Article in English | MEDLINE | ID: mdl-18003898

ABSTRACT

A heterozygous Ile4898 to Thr (I4898T) mutation in the human type 1 ryanodine receptor/Ca(2+) release channel (RyR1) leads to a severe form of central core disease. We created a mouse line in which the corresponding Ryr1(I4895T) mutation was introduced by using a "knockin" protocol. The heterozygote does not exhibit an overt disease phenotype, but homozygous (IT/IT) mice are paralyzed and die perinatally, apparently because of asphyxia. Histological analysis shows that IT/IT mice have greatly reduced and amorphous skeletal muscle. Myotubes are small, nuclei remain central, myofibrils are disarranged, and no cross striation is obvious. Many areas indicate probable degeneration, with shortened myotubes containing central stacks of pyknotic nuclei. Other manifestations of a delay in completion of late stages of embryogenesis include growth retardation and marked delay in ossification, dermatogenesis, and cardiovascular development. Electron microscopy of IT/IT muscle demonstrates appropriate targeting and positioning of RyR1 at triad junctions and a normal organization of dihydropyridine receptor (DHPR) complexes into RyR1-associated tetrads. Functional studies carried out in cultured IT/IT myotubes show that ligand-induced and DHPR-activated RyR1 Ca(2+) release is absent, although retrograde enhancement of DHPR Ca(2+) conductance is retained. IT/IT mice, in which RyR1-mediated Ca(2+) release is abolished without altering the formation of the junctional DHPR-RyR1 macromolecular complex, provide a valuable model for elucidation of the role of RyR1-mediated Ca(2+) signaling in mammalian embryogenesis.


Subject(s)
Calcium/metabolism , Homozygote , Ryanodine Receptor Calcium Release Channel/metabolism , Animals , Animals, Newborn , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Fetal Growth Retardation/genetics , Fetal Growth Retardation/metabolism , Fetal Growth Retardation/pathology , Gene Expression Regulation, Developmental , Heart/embryology , Isoleucine/genetics , Isoleucine/metabolism , Mice , Mice, Transgenic , Microscopy, Electron , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/ultrastructure , Muscle, Skeletal/embryology , Muscle, Skeletal/metabolism , Mutation/genetics , Ryanodine Receptor Calcium Release Channel/genetics , Skeleton , Threonine/genetics , Threonine/metabolism
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