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One Crystal,Two Temperatures: Cryocooling Penalties Alter Ligand Binding to Transient Protein Sites 下载免费PDF全文
Dr. Marcus Fischer Prof. Dr. Brian K. Shoichet Prof. Dr. James S. Fraser 《Chembiochem : a European journal of chemical biology》2015,16(11):1560-1564
Interrogating fragment libraries by X‐ray crystallography is a powerful strategy for discovering allosteric ligands for protein targets. Cryocooling of crystals should theoretically increase the fraction of occupied binding sites and decrease radiation damage. However, it might also perturb protein conformations that can be accessed at room temperature. Using data from crystals measured consecutively at room temperature and at cryogenic temperature, we found that transient binding sites could be abolished at the cryogenic temperatures employed by standard approaches. Changing the temperature at which the crystallographic data was collected could provide a deliberate perturbation to the equilibrium of protein conformations and help to visualize hidden sites with great potential to allosterically modulate protein function. 相似文献
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Emmanuel K. Toroitich Anthony M. Ciancone Heung Sik Hahm Skylar M. Brodowski Adam H. Libby Ku-Lung Hsu 《Chembiochem : a European journal of chemical biology》2021,22(12):2134-2139
Sulfonyl-triazoles have emerged as a new reactive group for covalent modification of tyrosine sites on proteins through sulfur-triazole exchange (SuTEx) chemistry. The extent to which this sulfur electrophile can be tuned for developing ligands with cellular activity remains largely underexplored. Here, we performed fragment-based ligand discovery in live cells to identify SuTEx compounds capable of liganding tyrosine sites on diverse protein targets. We verified our quantitative chemical proteomic findings by demonstrating concentration-dependent activity of SuTEx ligands, but not inactive counterparts, against recombinant protein targets directly in live cells. Our structure-activity relationship studies identified the SuTEx ligand HHS-0701 as a cell-active inhibitor capable of blocking prostaglandin reductase 2 (PTGR2) biochemical activity. 相似文献
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Dr. Andrea C. Kneuttinger Martin Winter Nadja A. Simeth Kristina Heyn Prof. Dr. Rainer Merkl Prof. Dr. Burkhard König Prof. Dr. Reinhard Sterner 《Chembiochem : a European journal of chemical biology》2018,19(16):1750-1757
The artificial regulation of proteins by light is an emerging subdiscipline of synthetic biology. Here, we used this concept to photocontrol both catalysis and allostery within the heterodimeric enzyme complex imidazole glycerol phosphate synthase (ImGP‐S). ImGP‐S consists of the cyclase subunit HisF and the glutaminase subunit HisH, which is allosterically stimulated by substrate binding to HisF. We show that a light‐sensitive diarylethene (1,2‐dithienylethene, DTE)‐based competitive inhibitor in its ring‐open state binds with low micromolar affinity to the cyclase subunit and displaces its substrate from the active site. As a consequence, catalysis by HisF and allosteric stimulation of HisH are impaired. Following UV‐light irradiation, the DTE ligand adopts its ring‐closed state and loses affinity for HisF, restoring activity and allostery. Our approach allows for the switching of ImGP‐S activity and allostery during catalysis and appears to be generally applicable for the light regulation of other multienzyme complexes. 相似文献
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Crystal Structure of Human Soluble Adenylate Cyclase Reveals a Distinct,Highly Flexible Allosteric Bicarbonate Binding Pocket 下载免费PDF全文
Susanne M. Saalau‐Bethell Dr. Valerio Berdini Dr. Anne Cleasby Dr. Miles Congreve Dr. Joseph E. Coyle Victoria Lock Dr. Christopher W. Murray Dr. M. Alistair O'Brien Sharna J. Rich Tracey Sambrook Dr. Mladen Vinkovic Dr. Jeff R. Yon Dr. Harren Jhoti 《ChemMedChem》2014,9(4):823-832
Soluble adenylate cyclases catalyse the synthesis of the second messenger cAMP through the cyclisation of ATP and are the only known enzymes to be directly activated by bicarbonate. Here, we report the first crystal structure of the human enzyme that reveals a pseudosymmetrical arrangement of two catalytic domains to produce a single competent active site and a novel discrete bicarbonate binding pocket. Crystal structures of the apo protein, the protein in complex with α,β‐methylene adenosine 5′‐triphosphate (AMPCPP) and calcium, with the allosteric activator bicarbonate, and also with a number of inhibitors identified using fragment screening, all show a flexible active site that undergoes significant conformational changes on binding of ligands. The resulting nanomolar‐potent inhibitors that were developed bind at both the substrate binding pocket and the allosteric site, and can be used as chemical probes to further elucidate the function of this protein. 相似文献
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Tammy Shim Jody Pacalon Won-Cheol Kim Xiaojing Cong Jrmie Topin Jrme Golebiowski Cheil Moon 《International journal of molecular sciences》2022,23(20)
Mammals recognize chemicals in the air via G protein-coupled odorant receptors (ORs). In addition to their orthosteric binding site, other segments of these receptors modulate ligand recognition. Focusing on human hOR1A1, which is considered prototypical of class II ORs, we used a combination of molecular modeling, site-directed mutagenesis, and in vitro functional assays. We showed that the third extracellular loop of ORs (ECL3) contributes to ligand recognition and receptor activation. Indeed, site-directed mutations in ECL3 showed differential effects on the potency and efficacy of both carvones, citronellol, and 2-nonanone. 相似文献
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Alzheimer’s disease (AD) is the most common cause of dementia, characterized by progressive cognitive decline and neurodegenerative disorder. Abnormal aggregations of intracellular neurofibrillary tangles (NFTs) and unusual accumulations of extracellular amyloid-β (Aβ) peptides are two important pathological features in AD brains. However, in spite of large-scale clinical studies and computational simulations, the molecular mechanisms of AD development and progression are still unclear. In this study, we divided all of the samples into two groups: early stage (Braak score I–III) and later stage (Braak score IV–VI). By big database mining, the candidate genetic and epigenetic networks (GEN) have been constructed. In order to find out the real GENs for two stages of AD, we performed systems identification and system order detection scheme to prune false positives with the help of corresponding microarray data. Applying the principal network projection (PNP) method, core GENs were extracted from real GENs based on the projection values. By the annotation of KEGG pathway, we could obtain core pathways from core GENs and investigate pathogenetic mechanisms for the early and later stage of AD, respectively. Consequently, according to pathogenetic mechanisms, several potential biomarkers are identified as drug targets for multiple-molecule drug design in the treatment of AD. 相似文献
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Ilham M. Alshiraihi Dillon K. Jarrell Zeyad Arhouma Kelly N. Hassell Jaelyn Montgomery Alyssa Padilla Hend M. Ibrahim Debbie C. Crans Takamitsu A. Kato Mark A. Brown 《International journal of molecular sciences》2020,21(24)
SMYD3 is a lysine methyltransferase that regulates the expression of over 80 genes and is required for the uncontrolled proliferation of most breast, colorectal, and hepatocellular carcinomas. The elimination of SMYD3 restores normal expression patterns of these genes and halts aberrant cell proliferation, making it a promising target for small molecule inhibition. In this study, we sought to establish a proof of concept for our in silico/in vitro hit-to-lead enzyme inhibitor development platform and to identify a lead small molecule candidate for SMYD3 inhibition. We used Schrodinger® software to screen libraries of small molecules in silico and the five compounds with the greatest predicted binding affinity within the SMYD3 binding pocket were purchased and assessed in vitro in direct binding assays and in breast cancer cell lines. We have confirmed the ability of one of these inhibitors, Inhibitor-4, to restore normal rates of cell proliferation, arrest the cell cycle, and induce apoptosis in breast cancer cells without affecting wildtype cell behavior. Our results provide a proof of concept for this fast and affordable small molecule hit-to-lead methodology as well as a promising candidate small molecule SMYD3 inhibitor for the treatment of human cancer. 相似文献
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Carina A. Sommer Dr. Andreas Eichinger Prof. Dr. Arne Skerra 《Chembiochem : a European journal of chemical biology》2020,21(4):469-472
Boronic acids have long been known to form cyclic diesters with cis-diol compounds, including many carbohydrates. This phenomenon was previously exploited to create an artificial lectin by incorporating p-borono-l -phenylalanine (Bpa) into the ligand pocket of an engineered lipocalin, resulting in a so-called Borocalin. Here we describe the X-ray analysis of its covalent complex with 4-nitrocatechol as a high-affinity model ligand. As expected, the crystal structure reveals the formation of a cyclic diester between the biosynthetic boronate side chain and the two ortho-hydroxy substituents of the benzene ring. Interestingly, the boron also has a hydroxide ion associated, despite an only moderately basic pH 8.5 in the crystallization buffer. The complex is stabilized by a polar contact to the side chain of Asn134 within the ligand pocket, thus validating the functional design of the Borocalin as an artificial sugar-binding protein. Our structural analysis demonstrates how a boronate can form a thermodynamically stable diester with a vicinal diol in a tetrahedral configuration in aqueous solution near physiological pH. Moreover, our data provide a basis for the further engineering of the Borocalin with the goal of specific recognition of biologically relevant glycans. 相似文献
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Matrix metalloproteinases (MMPs) are a class of zinc dependent endopeptidases which play a crucial role in a multitude of severe diseases such as cancer and osteoarthritis. We employed MMP-13 as the target enzyme for the structure-based design and synthesis of inhibitors able to recognize the catalytic zinc ion in addition to an allosteric binding site in order to increase the affinity of the ligand. Guided by molecular modeling, we optimized an initial allosteric inhibitor by addition of linker fragments and weak zinc binders for recognition of the catalytic center. Furthermore we improved the lipophilic ligand efficiency (LLE) of the initial inhibitor by adding appropriate zinc binding fragments to lower the clogP values of the inhibitors, while maintaining their potency. All synthesized inhibitors showed elevated affinity compared to the initial hit, also most of the novel inhibitors displayed better LLE. Derivatives with carboxylic acids as the zinc binding fragments turned out to be the most potent inhibitors (compound 3 (ZHAWOC5077): IC50 = 134 nM) whereas acyl sulfonamides showed the best lipophilic ligand efficiencies (compound 18 (ZHAWOC5135): LLE = 2.91). 相似文献
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Cytochrome P450 3A4 (CYP3A4) is a major human drug-metabolizing enzyme, notoriously known for its extreme substrate promiscuity, allosteric behavior, and implications in drug–drug interactions. Despite extensive investigations, the mechanism of ligand binding to CYP3A4 is not fully understood. We determined the crystal structure of CYP3A4 complexed with fluorol, a small fluorescent dye that can undergo hydroxylation. In the structure, fluorol associates to the substrate channel, well suited for the binding of planar polyaromatic molecules bearing polar groups, through which stabilizing H-bonds with the polar channel residues, such as Thr224 and Arg372, can be established. Mutagenesis, spectral, kinetic, and functional data confirmed the involvement but not strict requirement of Thr224 for the association of fluorol. Collectively, our data identify the substrate channel as a high-affinity ligand binding site and support the notion that hydrophobic ligands first dock to the nearby peripheral surface, before migrating to the channel and, subsequently, into the active site. 相似文献
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Structural Insight into the Allosteric Coupling of Cu1 Site and Trinuclear Cu Cluster in CotA Laccase 下载免费PDF全文
Dr. Tian Xie Dr. Zhongchuan Liu Prof. Dr. Ganggang Wang 《Chembiochem : a European journal of chemical biology》2018,19(14):1502-1506
In laccase, type 1 copper (Cu1) was connected to the trinuclear copper center (TNC) by the conserved Cys–His bridge. An allosteric coupling between the two redox sites has been reported; however, the molecular mechanism underlining the allosteric coupling is unknown. In this study, ligands of the two type 3 copper sites, including His491 and His493, in CotA were mutated to Cys or Ala. The crystal structures revealed that mutations at His491 and His493 caused rearrangement of the hydrogen‐bond network and geometric distortion of the TNC, which severely impaired the activities of mutants H493A, H493C, and H491C. In addition, the change in TNC affected hydrogen bonds around Cys492 in the mutants and led to Cu1 being partially reduced. These results not only decipher the mechanism of allosteric coupling between Cu1 and TNC in laccase, but also pave the way for laccase protein engineering. 相似文献
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Dr. Rhian S. Holvey Dr. Eugene Valkov Prof. David Neal Dr. Murray Stewart Prof. Chris Abell 《ChemMedChem》2015,10(7):1232-1239
Protein–protein interactions are difficult therapeutic targets, and inhibiting pathologically relevant interactions without disrupting other essential ones presents an additional challenge. Herein we report how this might be achieved for the potential anticancer target, the TPX2–importin‐α interaction. Importin‐α is a nuclear transport protein that regulates the spindle assembly protein TPX2. It has two binding sites—major and minor—to which partners bind. Most nuclear transport cargoes use the major site, whereas TPX2 binds principally to the minor site. Fragment‐based approaches were used to identify small molecules that bind importin‐α, and crystallographic studies identified a lead series that was observed to bind specifically to the minor site, representing the first ligands specific for this site. Structure‐guided synthesis informed the elaboration of these fragments to explore the source of ligand selectivity between the minor and major sites. These ligands are starting points for the development of inhibitors of this protein–protein interaction. 相似文献
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Discovery of Diverse Small‐Molecule Inhibitors of Mammalian Sterile20‐like Kinase 3 (MST3) 下载免费PDF全文
Dr. Sanne H. Olesen Dr. Jin‐Yi Zhu Dr. Mathew P. Martin Prof. Ernst Schönbrunn 《ChemMedChem》2016,11(11):1137-1144
Increasing evidence suggests key roles for members of the mammalian Sterile20‐like (MST) family of kinases in many aspects of biology. MST3 is a member of the STRIPAK complex, the deregulation of which has recently been associated with cancer cell migration and metastasis. Targeting MST3 with small‐molecule inhibitors may be beneficial for the treatment of certain cancers, but little information exists on the potential of kinase inhibitor scaffolds to engage with MST3. In this study we screened MST3 against a library of 277 kinase inhibitors using differential scanning fluorimetry and confirmed 14 previously unknown MST3 inhibitors by X‐ray crystallography. These compounds, of which eight are in clinical trials or FDA approved, comprise nine distinct chemical scaffolds that inhibit MST3 enzymatic activity with IC50 values between 0.003 and 23 μm . The structure–activity relationships explain the differential inhibitory activity of these compounds against MST3 and the structural basis for high binding potential, the information of which may serve as a framework for the rational design of MST3‐selective inhibitors as potential therapeutics and to interrogate the function of this enzyme in diseased cells. 相似文献
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Chisato Yoshikawa Hiroaki Ishida Nami Ohashi Toshimasa Itoh 《International journal of molecular sciences》2021,22(8)
Peroxisome proliferator-activated receptor γ (PPARγ) is a molecular target of metabolic syndrome and inflammatory disease. PPARγ is an important nuclear receptor and numerous PPARγ ligands were developed to date; thus, efficient assay methods are important. Here, we investigated the incorporation of 7-diethylamino coumarin into the PPARγ agonist rosiglitazone and used the compound in a binding assay for PPARγ. PPARγ-ligand-incorporated 7-methoxycoumarin, 1, showed weak fluorescence intensity in a previous report. We synthesized PPARγ-ligand-incorporating coumarin, 2, in this report, and it enhanced the fluorescence intensity. The PPARγ ligand 2 maintained the rosiglitazone activity. The obtained partial agonist 6 appeared to act through a novel mechanism. The fluorescence intensity of 2 and 6 increased by binding to the ligand binding domain (LBD) of PPARγ and the affinity of reported PPARγ ligands were evaluated using the probe. 相似文献
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Merging Allosteric and Active Site Binding Motifs: De novo Generation of Target Selectivity and Potency via Natural‐Product‐Derived Fragments 下载免费PDF全文
The de novo design of molecules from scratch with tailored biological activity is still the major intellectual challenge in chemical biology and drug discovery. Herein we validate natural‐product‐derived fragments (NPDFs) as excellent molecular seeds for the targeted de novo discovery of lead structures for the modulation of therapeutically relevant proteins. The application of this de novo approach delivered, in synergy with the combination of allosteric and active site binding motifs, highly selective and ligand‐efficient non‐zinc‐binding ( 3 : 4‐{[5‐(2‐{[(3‐methoxyphenyl)methyl]carbamoyl}eth‐1‐yn‐1‐yl)‐2,4‐dioxo‐1,2,3,4‐tetrahydropyrimidin‐1‐yl]methyl}benzoic acid) as well as zinc‐binding ( 4 : 4‐({5‐[2‐({[3‐(3‐carboxypropoxy)phenyl]methyl}carbamoyl)eth‐1‐yn‐1‐yl]‐2,4‐dioxo‐1,2,3,4‐tetrahydropyrimidin‐1‐yl}methyl)benzoic acid) uracil‐based MMP‐13 inhibitors presenting IC50 values of 11 nM ( 3 : LE=0.35) and 6 nM ( 4 : LE=0.31). 相似文献