scholarly journals Discovery of an IKK2 Site that Allosterically Controls Its Activation

2021 ◽  
Author(s):  
Sonjiala Jackson Hotchkiss ◽  
Maria Carmen Mulero ◽  
Garrett J. Chan ◽  
Tapan Biswas ◽  
Smarajit Polley ◽  
...  
Keyword(s):  
Small Molecule ◽  
Inflammatory Diseases ◽  
Binding Pocket ◽  
Allosteric Site ◽  
Allosteric Inhibitors ◽  
Allosteric Sites ◽  
Hydrogen Deuterium ◽  
Hdx Ms ◽  
Target Effects

ABSTRACTIκB kinase 2/β (IKK2) is a critical regulator of inflammation which is inducibly activated by a host of stimuli. Aberrant activation of IKK2 is the leading cause of most inflammatory diseases and many associated cancers. Efforts to prevent these diseases by small-molecule inhibitors of IKK2 activity have not been successful. Most inhibitors developed for IKK2 are ATP-competitive, and they are toxic in vivo due to their off-target effects. Here we focused on identifying inhibitors to block IKK2 activity from an allosteric site, not the ATP-binding pocket. Using virtual screening, we first identified several candidate allosteric sites and screened for potential small-molecule binders, and then selected candidates inhibitory to IKK2 activity using cell-based functional assays. Hydrogen deuterium exchange coupled to mass-spectrometry (HDX-MS) and MS-MS assays revealed that a class of benzoyl conjugates of pyrrolidinedione covalently bound to a site located at the interface of the kinase domain (KD) and the helical domain (SDD), and inhibited IKK2 activation allosterically by preventing phosphorylation of its activation loop serines. Additionally, this class of inhibitor partially blocks IKK2’s catalytic activity by enhancing dynamics within the ATP binding pocket and likely the general active site. Hydrogen deuterium exchange (HDX) experiments further revealed that while binding of substrate ATP perturbs only the local structure surrounding its binding site, binding to ATP-competitive or allosteric inhibitors induces structural perturbations in an expansive area including the helical domain. We propose that these allosteric sites can act as specific targets for the development of novel potent IKK inhibitors.SIGNIFICANCEAberrant activation of IKK2 is the leading cause of most inflammatory diseases and many associated cancers. Most inhibitors developed for IKK2 are ATP-competitive, and they are toxic in vivo due to their off-target effects. By combination of virtual screening and cell-based functional assays, we identified small-molecule binders of the class of benzoyl conjugates of pyrrolidinedione that block IKK2 activity from an allosteric site through covalent attachment and could be specific only for IKK2. HDX-MS and MS-MS assays identified a binding pocket with a ‘Cys-Cys motif’ for these inhibitors, and revealed specific differences in IKK2 dynamics upon binding to substrate ATP vs ATP-competitive and allosteric inhibitors. Present work provides a framework for the development of allosteric inhibitors to combat IKK2-induced diseases inhibitors.

scholarly journals New routes for PTP1B allosteric inhibition by multitemperature crystallography, fragment screening and covalent tethering

2017 ◽  
Author(s):  
Daniel A. Keedy ◽  
Zachary B. Hill ◽  
Justin T. Biel ◽  
Emily Kang ◽  
T. Justin Rettenmaier ◽  
...  
Keyword(s):  
Small Molecule ◽  
Tyrosine Phosphatase ◽  
Allosteric Site ◽  
Allosteric Inhibitors ◽  
Modulate Function ◽  
X Ray Crystallography ◽  
Fragment Screening ◽  
Inherent Feature ◽  
Allosteric Sites ◽  
Alternative Routes

AbstractAllostery is an inherent feature of proteins and provides alternative routes to regulating function. Small-molecule allosteric inhibitors are often desirable; however, it remains challenging to identify surface sites in proteins which can bind small molecules and modulate function. We identified new allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks. New modeling approaches reveal “hidden” low-occupancy conformational states for protein and ligands. Our results converge on a new allosteric site that is conformationally coupled to the active-site WPD loop, a hotspot for fragment binding, not conserved in the closest homolog, and distinct from other recently reported allosteric sites in PTP1B. Targeting this site with covalently tethered molecules allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure revealed by multitemperature crystallography can be exploited for developing allosteric modulators.

scholarly journals Recent Advances in the Discovery of CK2 Allosteric Inhibitors: From Traditional Screening to Structure-Based Design

Molecules ◽  
2020 ◽  
Vol 25 (4) ◽  
pp. 870 ◽  
Author(s):  
Xiaolan Chen ◽  
Chunqiong Li ◽  
Dada Wang ◽  
Yu Chen ◽  
Na Zhang
Keyword(s):  
Protein Kinase Ck2 ◽  
Binding Pocket ◽  
Atp Binding ◽  
Binding Modes ◽  
Allosteric Site ◽  
Allosteric Inhibitors ◽  
Atp Binding Site ◽  
Recent Advances ◽  
Allosteric Sites ◽  
Kinase Ck2

Protein kinase (CK2) has emerged as an attractive cancer therapeutic target and recent efforts have been made to develop its inhibitors. However, the development of selective inhibitors remains challenging because of the highly conserved ATP-binding pocket (orthosteric site) of kinase family. As an alternative strategy, allosteric inhibitors, by targeting the much more diversified allosteric site relative to the conserved ATP-binding site, achieve better pharmacological advantages than orthosteric inhibitors. Traditional serendipitous screening and structure-based design are robust tools for the discovery of CK2 allosteric inhibitors. In this review, we summarize the recent advances in the identification of CK2 allosteric inhibitors. Firstly, we briefly present the CK2 allosteric sites. Then, the allosteric inhibitors targeting the well-elucidated allosteric sites (α/β interface, αD pocket and interface between the Glycine-rich loop and αC-helix) are highlighted in the discovery process and possible binding modes with the allosteric sites are described. This study is expected to provide valuable clues for the design of CK2 allosteric inhibitors.

Approach in improving potency and selectivity of kinase inhibitors: Allosteric kinase inhibitors

2020 ◽  
Vol 21 ◽  
Author(s):  
Shangfei Wei ◽  
Tianming Zhao ◽  
Jie Wang ◽  
Xin Zhai
Keyword(s):  
Protein Interactions ◽  
Kinase Inhibitors ◽  
Binding Modes ◽  
Allosteric Inhibitors ◽  
Wide Range ◽  
Allosteric Sites ◽  
Protein Functions ◽  
Regulate Protein

: Allostery is an efficient and particular regulatory mechanism to regulate protein functions. Different from conserved orthosteric sites, allosteric sites have distinctive functional mechanism to form the complex regulatory network. In drug discovery, kinase inhibitors targeting the allosteric pockets have received extensive attention for the advantages of high selectivity and low toxicity. The approval of trametinib as the first allosteric inhibitor validated that allosteric inhibitors could be used as effective therapeutic drugs for treatment of diseases. To date, a wide range of allosteric inhibitors have been identified. In this perspective, we outline different binding modes and potential advantages of allosteric inhibitors. In the meantime, the research processes of typical and novel allosteric inhibitors are described briefly in terms of structureactivity relationships, ligand-protein interactions and in vitro and in vivo activity. Additionally, challenges as well as opportunities are presented.

scholarly journals Engineered anti-inflammatory peptides inspired by mapping an evasin–chemokine interaction

2020 ◽  
Vol 295 (32) ◽  
pp. 10926-10939 ◽  
Author(s):  
Benoit Darlot ◽  
James R. O. Eaton ◽  
Lucia Geis-Asteggiante ◽  
Gopala K. Yakala ◽  
Kalimuthu Karuppanan ◽  
...  
Keyword(s):  
Inflammatory Diseases ◽  
Titration Calorimetry ◽  
Protein Activity ◽  
Binding Interface ◽  
Chemokine Ligand ◽  
Therapeutic Development ◽  
Hydrogen Deuterium ◽  
Anti Inflammatory

Chemokines mediate leukocyte migration and homeostasis and are key targets in inflammatory diseases including atherosclerosis, cytokine storm, and chronic autoimmune disease. Chemokine redundancy and ensuing network robustness has frustrated therapeutic development. Salivary evasins from ticks bind multiple chemokines to overcome redundancy and are effective in several preclinical disease models. Their clinical development has not progressed because of concerns regarding potential immunogenicity, parenteral delivery, and cost. Peptides mimicking protein activity can overcome the perceived limitations of therapeutic proteins. Here we show that peptides possessing multiple chemokine-binding and anti-inflammatory activities can be developed from the chemokine-binding site of an evasin. We used hydrogen–deuterium exchange MS to map the binding interface of the evasin P672 that physically interacts with C–C motif chemokine ligand (CCL) 8 and synthesized a 16-mer peptide (BK1.1) based on this interface region in evasin P672. Fluorescent polarization and native MS approaches showed that BK1.1 binds CCL8, CCL7, and CCL18 and disrupts CCL8 homodimerization. We show that a BK1.1 derivative, BK1.3, has substantially improved ability to disrupt P672 binding to CCL8, CCL2, and CCL3 in an AlphaScreen assay. Using isothermal titration calorimetry, we show that BK1.3 directly binds CCL8. BK1.3 also has substantially improved ability to inhibit CCL8, CCL7, CCL2, and CCL3 chemotactic function in vitro. We show that local as well as systemic administration of BK1.3 potently blocks inflammation in vivo. Identification and characterization of the chemokine-binding interface of evasins could thus inspire the development of novel anti-inflammatory peptides that therapeutically target the chemokine network in inflammatory diseases.

scholarly journals An allosteric site on MKP5 reveals a strategy for small-molecule inhibition

2020 ◽  
Vol 13 (646) ◽  
pp. eaba3043 ◽  
Author(s):  
Zachary T. K. Gannam ◽  
Kisuk Min ◽  
Shanelle R. Shillingford ◽  
Lei Zhang ◽  
James Herrington ◽  
...  
Keyword(s):  
Small Molecule ◽  
Binding Pocket ◽  
Muscle Disease ◽  
Mitogen Activated Protein Kinase ◽  
Allosteric Site ◽  
Dystrophic Muscle ◽  
Smad2 Phosphorylation ◽  
Mitogen Activated Protein ◽  
P38α Mapk ◽  
Tgf Β1

The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) have been considered “undruggable,” but their position as regulators of the MAPKs makes them promising therapeutic targets. MKP5 has been suggested as a potential target for the treatment of dystrophic muscle disease. Here, we identified an inhibitor of MKP5 using a p38α MAPK–derived, phosphopeptide-based small-molecule screen. We solved the structure of MKP5 in complex with this inhibitor, which revealed a previously undescribed allosteric binding pocket. Binding of the inhibitor to this pocket collapsed the MKP5 active site and was predicted to limit MAPK binding. Treatment with the inhibitor recapitulated the phenotype of MKP5 deficiency, resulting in activation of p38 MAPK and JNK. We demonstrated that MKP5 was required for TGF-β1 signaling in muscle and that the inhibitor blocked TGF-β1–mediated Smad2 phosphorylation. TGF-β1 pathway antagonism has been proposed for the treatment of dystrophic muscle disease. Thus, allosteric inhibition of MKP5 represents a therapeutic strategy against dystrophic muscle disease.

The Design and Synthesis of Small Molecule Inhibitors of Collagen Binding to Integrin α2β1 as Antithrombotic Agents.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 306-306
Author(s):  
Meredith W. Miller ◽  
Soni Basra ◽  
Paul C. Billings ◽  
Jamie Gewirtz ◽  
William F. DeGrado ◽  
...  
Keyword(s):  
Small Molecule ◽  
Platelet Adhesion ◽  
Small Molecule Inhibitors ◽  
Critical Event ◽  
Platelet Activity ◽  
Allosteric Site ◽  
Collagen Binding ◽  
Glycoprotein Vi

Abstract Vascular damage due to trauma or disease exposes circulating platelets to collagen in the subendothelial matrix. This is a critical event in the formation of a hemostatic plug or an occluding thrombus because collagen is not only a substrate for platelet adhesion but is also a strong platelet agonist. Platelets possess two physiologic collagen receptors: glycoprotein VI, a member of the immunoglobin superfamily, and the integrin α2β1. To design small molecule inhibitors of the interaction of platelets with collagen, we focused on α2β1 as a target because murine models of α2β1 deficiency display normal bleeding times and only a slight decrease in platelet activation by collagen and because the small number of reported patients with congenital α2β1 deficiency demonstrated only a mild bleeding diathesis. Thus, α2β1 antagonists could be effective anti-thrombotic agents with minimal toxicity, especially when combined with other anti-platelet drugs. We have developed a class of compounds that target the I-like domain of the β1 subunit, an allosteric site that regulates collagen binding to α2β1 by preventing the conversion of α2β1 from an inactive (low affinity) to an active (high affinity) conformation. This class of compounds is based on a proline-substituted 2,3-diaminopropionic acid scaffold. Structure-activity relationship studies of the scaffold have focused on optimization of the proline moiety, the urea functionality, and the sulfonyl group and have resulted in the development of potent inhibitors of α2β1-mediated platelet adhesion to collagen with IC50’s in the high picomolar to low nanomolar range. In particular, optimization of the proline moiety lead to compounds with high potency: transitioning from proline (DB496, IC50 of 29–62 nM) to a thiazolidine (SB68A) improved the IC50 to 2–8 nM; adding a methyl group at the 2 position of the thiazolidine (SB68B) slightly improved the IC50 to 1–12 nM; adding two methyl groups at the 5 position of the thiazolidine (SW4-161) resulted in a lead compound with an IC50 of 0.33–8 nM. As expected, the compounds had no effect on the binding of isolated α2 I-domains to collagen, consistent with their I-like domain mode of activity. Further, they were specific for α2β1-mediated platelet adhesion to collagen because they had no impact on ADP-stimulated platelet aggregation when added at 2 μM, a concentration more than 100-fold greater than the IC50 for inhibition of platelet adhesion to collagen. The compounds were also strong inhibitors of murine platelet adhesion to collagen and when tested in the ferric chloride-initiated murine carotid artery injury model, displayed activity similar to aspirin. Thus, 71% of untreated mice in this thrombosis model developed occlusive thrombi that remained stable for the 30 min duration of the assay, whereas stable thrombi developed in only 32% of mice treated with 1g/kg aspirin orally and in 41% of mice receiving 60 mg/kg CSW4-161intravenously. In summary, we have developed a class of potent inhibitors of the integrin α2β1 that demonstrate both in vitro and in vivo anti-platelet activity. Further development of this class of compounds may result in novel and relatively non-toxic anti-thrombotic agents.

scholarly journals Two helices control the dynamic crosstalk between the catalytic domains of LRRK2

2021 ◽  
Author(s):  
Jui-Hung Weng ◽  
Phillop C. Aoto ◽  
Robin Lorenz ◽  
Jian Wu ◽  
Sven H. Schmidt ◽  
...  
Keyword(s):  
Md Simulations ◽  
Kinase Domain ◽  
Pharmacological Intervention ◽  
Exchange Mass Spectrometry ◽  
Allosteric Sites ◽  
Hydrogen Deuterium ◽  
Wd40 Domain ◽  
Deuterium Exchange Mass Spectrometry ◽  
And Function ◽  
Hdx Ms

The two major molecular switches in biology, kinases and GTPases, are both contained in the Parkinson’s Disease-related Leucine-rich repeat kinase 2 (LRRK2). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Molecular Dynamics (MD) simulations, we generated a comprehensive dynamic allosteric portrait of the C-terminal domains of LRRK2 (LRRK2 RCKW). We identified two helices that shield the kinase domain and regulate LRRK2 conformation and function. One docking helix in COR-B (Dk-Helix) tethers the COR-B domain to the αC helix of the kinase domain and faces its Activation Loop, while the C-terminal helix (Ct-Helix) extends from the WD40 domain and interacts with both kinase lobes. The Ct-Helix and the N-terminus of the Dk-Helix create a “cap” that regulates the N-Lobe of the kinase domain. Our analyses reveal allosteric sites for pharmacological intervention and confirm the kinase domain as the central hub for conformational control.

scholarly journals Identification and Biological Evaluation of CK2 Allosteric Fragments through Structure-Based Virtual Screening

Molecules ◽  
2020 ◽  
Vol 25 (1) ◽  
pp. 237 ◽  
Author(s):  
Chunqiong Li ◽  
Xuewen Zhang ◽  
Na Zhang ◽  
Yue Zhou ◽  
Guohui Sun ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Interaction Mechanism ◽  
Pharmacophore Model ◽  
Binding Pocket ◽  
Biological Evaluation ◽  
Atp Binding ◽  
Allosteric Site ◽  
Allosteric Inhibitors ◽  
Competitive Inhibitors ◽  
Reduced Toxicity

Casein kinase II (CK2) is considered as an attractive cancer therapeutic target, and recent efforts have been made to develop its ATP-competitive inhibitors. However, achieving selectivity with respect to related kinases remains challenging due to the highly conserved ATP-binding pocket of kinases. Allosteric inhibitors, by targeting the much more diversified allosteric site relative to the highly conserved ATP-binding pocket, might be a promising strategy with the enhanced selectivity and reduced toxicity than ATP-competitive inhibitors. The previous studies have highlighted the traditional serendipitousity of discovering allosteric inhibitors owing to the complicate allosteric modulation. In this current study, we identified the novel allosteric inhibitors of CK2α by combing structure-based virtual screening and biological evaluation methods. The structure-based pharmacophore model was built based on the crystal structure of CK2α-compound 15 complex. The ChemBridge fragment library was searched by evaluating the fit values of these molecules with the optimized pharmacophore model, as well as the binding affinity of the CK2α-ligand complexes predicted by Alloscore web server. Six hits forming the holistic interaction mechanism with the αD pocket were retained after pharmacophore- and Alloscore-based screening for biological test. Compound 3 was found to be the most potent non-ATP competitive CK2α inhibitor (IC50 = 13.0 μM) with the anti-proliferative activity on A549 cancer cells (IC50 = 23.1 μM). Our results provide new clues for further development of CK2 allosteric inhibitors as anti-cancer hits.

scholarly journals Design and Development of Novel Urea, Sulfonyltriurea, and Sulfonamide Derivatives as Potential Inhibitors of Sphingosine Kinase 1

2020 ◽  
Vol 13 (6) ◽  
pp. 118
Author(s):  
Sonam Roy ◽  
Amarjyoti Das Mahapatra ◽  
Taj Mohammad ◽  
Preeti Gupta ◽  
Mohamed F. Alajmi ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Drug Targets ◽  
Inflammatory Diseases ◽  
Cancer Therapeutics ◽  
Sphingosine Kinase ◽  
Binding Pocket ◽  
Van Der Waals Interactions ◽  
Titration Calorimetry ◽  
Sphingosine Kinase 1

Sphingosine kinase 1 (SphK1) is one of the well-studied drug targets for cancer and inflammatory diseases. Recently discovered small-molecule inhibitors of SphK1 have been recommended in cancer therapeutics; however, selectivity and potency of first-generation inhibitors are great challenge. In search of effective SphK1 inhibitors, a set of small molecules have been designed and synthesized bearing urea, sulfonylurea, sulfonamide, and sulfonyltriurea groups. The binding affinity of these inhibitors was measured by fluorescence-binding assay and isothermal titration calorimetry. Compounds 1, 5, 6, and 7 showed an admirable binding affinity to the SphK1 in the sub-micromolar range and significantly inhibited SphK1 activity with admirable IC50 values. Molecular docking studies revealed that these compounds fit well into the sphingosine binding pocket of SphK1 and formed significant number of hydrogen bonds and van der Waals interactions. These molecules may be exploited as potent and selective inhibitors of SphK1 that could be implicated in cancer therapeutics after the required in vivo validation.

scholarly journals Combining small-molecule bioconjugation and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to expose allostery: the case of human cytochrome P450 3A4

2020 ◽  
Author(s):  
Julie Ducharme ◽  
Christopher J. Thibodeaux ◽  
Karine Auclair
Keyword(s):  
Mass Spectrometry ◽  
Cytochrome P450 ◽  
Cytochrome P450 3A4 ◽  
Hydrogen Deuterium Exchange ◽  
Allosteric Site ◽  
Exchange Mass Spectrometry ◽  
Human Cytochrome ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

AbstractWe report herein a novel approach to study allostery which combines the use of carefully selected bioconjugates and hydrogen-deuterium exchange mass spectrometry (HDX-MS). The utility of our method is demonstrated using human cytochrome P450 3A4 (CYP3A4). CYP3A4 is arguably the most important drug-metabolizing enzyme, and as such, is involved in numerous drug interactions. Diverse allosteric ligand effects have been reported for this enzyme, yet the structural mechanism of these phenomena remain poorly understood. We have described different CYP3A4-effector bioconjugates, some of which mimic the allosteric effect of positive effectors on CYP3A4, while others show activity enhancement even though the label does not occupy the allosteric pocket (agonistic), or do not show activation while still blocking the allosteric site (antagonistic). These bioonjugates were studied here by HDX-MS, which enabled us to better define the position of the allosteric site, and to identify important regions involved in CYP3A4 activation.

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