Publications by authors named "Simin Rahighi"

15 Publications

  • Page 1 of 1

Cyclic Peptides as Protein Kinase Inhibitors: Structure-Activity Relationship and Molecular Modeling.

J Chem Inf Model 2021 06 17;61(6):3015-3026. Epub 2021 May 17.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, California 92618, United States.

Under-expression or overexpression of protein kinases has been shown to be associated with unregulated cell signal transduction in cancer cells. Therefore, there is major interest in designing protein kinase inhibitors as anticancer agents. We have previously reported [WR], a peptide containing alternative arginine (R) and tryptophan (W) residues as a non-competitive c-Src tyrosine kinase inhibitor. A number of larger cyclic peptides containing alternative hydrophobic and positively charged residues [WR] ( = 6-9) and hybrid cyclic-linear peptides, [RK]W and [RK]W, containing R and W residues were evaluated for their protein kinase inhibitory potency. Among all the peptides, cyclic peptide [WR] was found to be the most potent tyrosine kinase inhibitor. [WR] showed higher inhibitory activity (IC = 0.21 μM) than [WR], [WR], [WR], and [WR] with IC values of 0.81, 0.57, 0.35, and 0.33 μM, respectively, against c-Src kinase as determined by a radioactive assay using [γ-P]ATP. Consistent with the result above, [WR] inhibited other protein kinases such as Abl kinase activity with an IC value of 0.35 μM, showing 2.2-fold higher inhibition than [WR] (IC = 0.79 μM). [WR] also inhibited PKCa kinase activity with an IC value of 2.86 μM, approximately threefold higher inhibition than [WR] (IC = 8.52 μM). A similar pattern was observed against Braf, c-Src, Cdk2/cyclin A1, and Lck. [WR] exhibited IC values of <0.25 μM against Akt1, Alk, and Btk. These data suggest that [WR] is consistently more potent than other cyclic peptides with a smaller ring size and hybrid cyclic-linear peptides [RK]W and [RK]W against selected protein kinases. Thus, the presence of R and W residues in the ring, ring size, and the number of amino acids in the structure of the cyclic peptide were found to be critical in protein kinase inhibitory potency. We identified three putative binding pockets through automated blind docking of cyclic peptides [WR]. The most populated pocket is located between the SH2, SH3, and N-lobe domains on the opposite side of the ATP binding site. The second putative pocket is formed by the same domains and located on the ATP binding site side of the protein. Finally, a third pocket was identified between the SH2 and SH3 domains. These results are consistent with the non-competitive nature of the inhibition displayed by these molecules. Molecular dynamics simulations of the protein-peptide complexes indicate that the presence of either [WR] or [WR] affects the plasticity of the protein and in particular the volume of the ATP binding site pocket in different ways. These results suggest that the second pocket is most likely the site where these peptides bind and offer a plausible rationale for the increased affinity of [WR].
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http://dx.doi.org/10.1021/acs.jcim.1c00320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8238896PMC
June 2021

Hydrophobic interactions between the HA helix and S4-S5 linker modulate apparent Ca sensitivity of SK2 channels.

Acta Physiol (Oxf) 2021 01 10;231(1):e13552. Epub 2020 Sep 10.

Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Irvine, CA, USA.

Aim: Small-conductance Ca -activated potassium (SK) channels are activated exclusively by increases in intracellular Ca that binds to calmodulin constitutively associated with the channel. Wild-type SK2 channels are activated by Ca with an EC value of ~0.3 μmol/L. Here, we investigate hydrophobic interactions between the HA helix and the S4-S5 linker as a major determinant of channel apparent Ca sensitivity.

Methods: Site-directed mutagenesis, electrophysiological recordings and molecular dynamic (MD) simulations were utilized.

Results: Mutations that decrease hydrophobicity at the HA-S4-S5 interface lead to Ca hyposensitivity of SK2 channels. Mutations that increase hydrophobicity result in hypersensitivity to Ca . The Ca hypersensitivity of the V407F mutant relies on the interaction of the cognate phenylalanine with the S4-S5 linker in the SK2 channel. Replacing the S4-S5 linker of the SK2 channel with the S4-S5 linker of the SK4 channel results in loss of the hypersensitivity caused by V407F. This difference between the S4-S5 linkers of SK2 and SK4 channels can be partially attributed to I295 equivalent to a valine in the SK4 channel. A N293A mutation in the S4-S5 linker also increases hydrophobicity at the HA-S4-S5 interface and elevates the channel apparent Ca sensitivity. The double N293A/V407F mutations generate a highly Ca sensitive channel, with an EC of 0.02 μmol/L. The MD simulations of this double-mutant channel revealed a larger channel cytoplasmic gate.

Conclusion: The electrophysiological data and MD simulations collectively suggest a crucial role of the interactions between the HA helix and S4-S5 linker in the apparent Ca sensitivity of SK2 channels.
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http://dx.doi.org/10.1111/apha.13552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736289PMC
January 2021

Discovery of Protein-Protein Interaction Inhibitors by Integrating Protein Engineering and Chemical Screening Platforms.

Cell Chem Biol 2020 11 28;27(11):1441-1451.e7. Epub 2020 Jul 28.

Institute of Biochemistry II, Goethe University, Frankfurt am Main, Germany; Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Branch for Translational Medicine and Pharmacology, Theodor-Stern-Kai 7, 60596 Frankfurt am Main, Germany; Buchmann Institute for Molecular Life Sciences, Frankfurt am Main, Germany. Electronic address:

Protein-protein interactions (PPIs) govern intracellular life, and identification of PPI inhibitors is challenging. Roadblocks in assay development stemming from weak binding affinities of natural PPIs impede progress in this field. We postulated that enhancing binding affinity of natural PPIs via protein engineering will aid assay development and hit discovery. This proof-of-principle study targets PPI between linear ubiquitin chains and NEMO UBAN domain, which activates NF-κB signaling. Using phage display, we generated ubiquitin variants that bind to the functional UBAN epitope with high affinity, act as competitive inhibitors, and structurally maintain the existing PPI interface. When utilized in assay development, variants enable generation of robust cell-based assays for chemical screening. Top compounds identified using this approach directly bind to UBAN and dampen NF-κB signaling. This study illustrates advantages of integrating protein engineering and chemical screening in hit identification, a development that we anticipate will have wide application in drug discovery.
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http://dx.doi.org/10.1016/j.chembiol.2020.07.010DOI Listing
November 2020

Molecular Recognition of M1-Linked Ubiquitin Chains by Native and Phosphorylated UBAN Domains.

J Mol Biol 2019 08 24;431(17):3146-3156. Epub 2019 Jun 24.

Chapman University School of Pharmacy (CUSP), Harry and Diane Rinker Health Science Campus, Chapman University, Irvine, CA 92618, USA. Electronic address:

Although the Ub-binding domain in ABIN proteins and NEMO (UBAN) is highly conserved, UBAN-containing proteins exhibit different Ub-binding properties, resulting in their diverse biological roles. Post-translational modifications further control UBAN domain specificity for poly-Ub chains. However, precisely, how the UBAN domain structurally confers such functional diversity remains poorly understood. Here we report crystal structures of ABIN-1 alone and in complex with one or two M1-linked di-Ub chains. ABIN-1 UBAN forms a homo-dimer that provides two symmetrical Ub-binding sites on either side of the coiled-coil structure. Moreover, crystal structures of ABIN1 UBAN in complex with di-Ub chains reveal a concentration-dependency of UBAN/di-Ub binding stoichiometry. Analysis of UBAN/M1-linked di-Ub binding characteristics indicates that phosphorylated S473 in OPTN and its corresponding phospho-mimetic residue in ABIN-1 (E484) are essential for high affinity interactions with M1-linked Ub chains. Also, a phospho-mimetic mutation of A303 in NEMO, corresponding to S473 of OPTN, increases binding affinity for M1-linked Ub chains. These findings are in line with the diverse physiological roles of UBAN domains, as phosphorylation of OPTN UBAN is required to enhance its binding to Ub during mitophagy.
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http://dx.doi.org/10.1016/j.jmb.2019.06.012DOI Listing
August 2019

Small-Molecule Activators of Glucose-6-phosphate Dehydrogenase (G6PD) Bridging the Dimer Interface.

ChemMedChem 2019 07 27;14(14):1321-1324. Epub 2019 Jun 27.

Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, 94305, USA.

We recently identified AG1, a small-molecule activator that functions by promoting oligomerization of glucose-6-phosphate dehydrogenase (G6PD) to the catalytically competent forms. Biochemical experiments indicate that the activation of G6PD by the original hit molecule (AG1) is noncovalent and that one C -symmetric region of the G6PD homodimer is important for ligand function. Consequently, the disulfide in AG1 is not required for activation of G6PD, and a number of analogues were prepared without this reactive moiety. Our study supports a mechanism of action whereby AG1 bridges the dimer interface at the structural nicotinamide adenine dinucleotide phosphate (NADP ) binding sites of two interacting G6PD monomers. Small molecules that promote G6PD oligomerization have the potential to provide a first-in-class treatment for G6PD deficiency. This general strategy could be applied to other enzyme deficiencies in which control of oligomerization can enhance enzymatic activity and/or stability.
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http://dx.doi.org/10.1002/cmdc.201900341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6701841PMC
July 2019

Correcting glucose-6-phosphate dehydrogenase deficiency with a small-molecule activator.

Nat Commun 2018 10 2;9(1):4045. Epub 2018 Oct 2.

Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, 94305, USA.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common human genetic enzymopathies, is caused by over 160 different point mutations and contributes to the severity of many acute and chronic diseases associated with oxidative stress, including hemolytic anemia and bilirubin-induced neurological damage particularly in newborns. As no medications are available to treat G6PD deficiency, here we seek to identify a small molecule that corrects it. Crystallographic study and mutagenesis analysis identify the structural and functional defect of one common mutant (Canton, R459L). Using high-throughput screening, we subsequently identify AG1, a small molecule that increases the activity of the wild-type, the Canton mutant and several other common G6PD mutants. AG1 reduces oxidative stress in cells and zebrafish. Furthermore, AG1 decreases chloroquine- or diamide-induced oxidative stress in human erythrocytes. Our study suggests that a pharmacological agent, of which AG1 may be a lead, will likely alleviate the challenges associated with G6PD deficiency.
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http://dx.doi.org/10.1038/s41467-018-06447-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168459PMC
October 2018

Linear ubiquitin chain-binding domains.

FEBS J 2018 08 30;285(15):2746-2761. Epub 2018 Apr 30.

Institute of Molecular Biotechnology (IMBA), Vienna Biocenter (VBC), Austria.

Ubiquitin modification (ubiquitination) of target proteins can vary with respect to chain lengths, linkage type, and chain forms, such as homologous, mixed, and branched ubiquitin chains. Thus, ubiquitination can generate multiple unique surfaces on a target protein substrate. Ubiquitin-binding domains (UBDs) recognize ubiquitinated substrates, by specifically binding to these unique surfaces, modulate the formation of cellular signaling complexes and regulate downstream signaling cascades. Among the eight different homotypic chain types, Met1-linked (also termed linear) chains are the only chains in which linkage occurs on a non-Lys residue of ubiquitin. Linear ubiquitin chains have been implicated in immune responses, cell death and autophagy, and several UBDs - specific for linear ubiquitin chains - have been identified. In this review, we describe the main principles of ubiquitin recognition by UBDs, focusing on linear ubiquitin chains and their roles in biology.
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http://dx.doi.org/10.1111/febs.14478DOI Listing
August 2018

Selective Binding of AIRAPL Tandem UIMs to Lys48-Linked Tri-Ubiquitin Chains.

Structure 2016 Mar 11;24(3):412-22. Epub 2016 Feb 11.

Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Ibaraki 305-0801, Japan; Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA; Structural Molecular Biology, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, USA; Photon Science, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, USA. Electronic address:

Lys48-linked ubiquitin chains act as the main targeting signals for protein degradation by the proteasome. Here we report selective binding of AIRAPL, a protein that associates with the proteasome upon exposure to arsenite, to Lys48-linked tri-ubiquitin chains. AIRAPL comprises two ubiquitin-interacting motifs in tandem (tUIMs) that are linked through a flexible inter-UIM region. In the complex crystal structure UIM1 binds the proximal ubiquitin, whereas UIM2 (the double-sided UIM) binds non-symmetrically to the middle and distal ubiquitin moieties on either side of the helix. Specificity of AIRAPL for Lys48-linked ubiquitin chains is determined by UIM2, and the flexible inter-UIM linker increases avidity by placing the two UIMs in an orientation that facilitates binding of the third ubiquitin to UIM1. Unlike middle and proximal ubiquitins, distal ubiquitin binds UIM2 through a novel surface, which leaves the Ile44 hydrophobic patch accessible for binding to the proteasomal ubiquitin receptors.
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http://dx.doi.org/10.1016/j.str.2015.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4775417PMC
March 2016

Crystal structure of a PCP/Sfp complex reveals the structural basis for carrier protein posttranslational modification.

Chem Biol 2014 Apr 3;21(4):552-562. Epub 2014 Apr 3.

Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt/Main, Max-von-Laue-Strasse 9, 60438 Frankfurt, Germany; Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt/Main, Max-von-Laue-Strasse 15, 60438 Frankfurt, Germany. Electronic address:

Phosphopantetheine transferases represent a class of enzymes found throughout all forms of life. From a structural point of view, they are subdivided into three groups, with transferases from group II being the most widespread. They are required for the posttranslational modification of carrier proteins involved in diverse metabolic pathways. We determined the crystal structure of the group II phosphopantetheine transferase Sfp from Bacillus in complex with a substrate carrier protein in the presence of coenzyme A and magnesium, and observed two protein-protein interaction sites. Mutational analysis showed that only the hydrophobic contacts between the carrier protein's second helix and the C-terminal domain of Sfp are essential for their productive interaction. Comparison with a similar structure of a complex of human proteins suggests that the mode of interaction is highly conserved in all domains of life.
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http://dx.doi.org/10.1016/j.chembiol.2014.02.014DOI Listing
April 2014

Mechanism underlying IκB kinase activation mediated by the linear ubiquitin chain assembly complex.

Mol Cell Biol 2014 Apr 27;34(7):1322-35. Epub 2014 Jan 27.

Cell Biology and Metabolism Group, Graduate School of Frontier Biosciences, Osaka University, Suita, Japan.

The linear ubiquitin chain assembly complex (LUBAC) ligase, consisting of HOIL-1L, HOIP, and SHARPIN, specifically generates linear polyubiquitin chains. LUBAC-mediated linear polyubiquitination has been implicated in NF-κB activation. NEMO, a component of the IκB kinase (IKK) complex, is a substrate of LUBAC, but the precise molecular mechanism underlying linear chain-mediated NF-κB activation has not been fully elucidated. Here, we demonstrate that linearly polyubiquitinated NEMO activates IKK more potently than unanchored linear chains. In mutational analyses based on the crystal structure of the complex between the HOIP NZF1 and NEMO CC2-LZ domains, which are involved in the HOIP-NEMO interaction, NEMO mutations that impaired linear ubiquitin recognition activity and prevented recognition by LUBAC synergistically suppressed signal-induced NF-κB activation. HOIP NZF1 bound to NEMO and ubiquitin simultaneously, and HOIP NZF1 mutants defective in interaction with either NEMO or ubiquitin could not restore signal-induced NF-κB activation. Furthermore, linear chain-mediated activation of IKK2 involved homotypic interaction of the IKK2 kinase domain. Collectively, these results demonstrate that linear polyubiquitination of NEMO plays crucial roles in IKK activation and that this modification involves the HOIP NZF1 domain and recognition of NEMO-conjugated linear ubiquitin chains by NEMO on another IKK complex.
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http://dx.doi.org/10.1128/MCB.01538-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3993567PMC
April 2014

Selectivity of the ubiquitin-binding modules.

FEBS Lett 2012 Aug 5;586(17):2705-10. Epub 2012 May 5.

Buchmann Institute for Molecular Life Sciences and Institute of Biochemistry II, Goethe University School of Medicine, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany.

Ubiquitin-binding modules are constituents of cellular proteins that mediate the effects of ubiquitylation by making transient, non-covalent interactions with ubiquitin molecules. While some ubiquitin-binding modules bind single ubiquitin moieties, others are selective for specific ubiquitin chains of different linkage types and lengths. In recent years, functions of ubiquitin chains that are polymerized through their Lys or N-terminal Met (i.e. linear chains) residues have been linked to a variety of cellular processes. Selectivity of ubiquitin-binding modules for different ubiquitin chain types appears as a key to the distinct regulatory consequences during protein quality control pathways, receptor endocytosis, gene transcription, signaling via the NF-κB pathway, and autophagy.
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http://dx.doi.org/10.1016/j.febslet.2012.04.053DOI Listing
August 2012

Conformational flexibility and rotation of the RING domain in activation of cullin-RING ligases.

Nat Struct Mol Biol 2011 Aug 3;18(8):863-5. Epub 2011 Aug 3.

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http://dx.doi.org/10.1038/nsmb.2117DOI Listing
August 2011

Selective binding of linear ubiquitin chains to NEMO in NF-kappaB activation.

Adv Exp Med Biol 2011 ;691:107-14

Institute of Biochemistry II and Cluster of Excellence Frankfurt, Goethe University School of Medicine, Theodor-Stern-Kai 7, D-60590 Frankfurt(Main), Germany.

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http://dx.doi.org/10.1007/978-1-4419-6612-4_11DOI Listing
April 2011

Unusual antibacterial property of mesoporous titania films: drastic improvement by controlling surface area and crystallinity.

Chem Asian J 2010 Sep;5(9):1978-83

World Premier International (WPI) Research Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki, 305-0044, Japan.

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http://dx.doi.org/10.1002/asia.201000351DOI Listing
September 2010

Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation.

Cell 2009 Mar;136(6):1098-109

Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Ibaraki, Japan.

Activation of nuclear factor-kappaB (NF-kappaB), a key mediator of inducible transcription in immunity, requires binding of NF-kappaB essential modulator (NEMO) to ubiquitinated substrates. Here, we report that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO selectively binds linear (head-to-tail) ubiquitin chains. Crystal structures of the UBAN motif revealed a parallel coiled-coil dimer that formed a heterotetrameric complex with two linear diubiquitin molecules. The UBAN dimer contacted all four ubiquitin moieties, and the integrity of each binding site was required for efficient NF-kappaB activation. Binding occurred via a surface on the proximal ubiquitin moiety and the canonical Ile44 surface on the distal one, thereby providing specificity for linear chain recognition. Residues of NEMO involved in binding linear ubiquitin chains are required for NF-kappaB activation by TNF-alpha and other agonists, providing an explanation for the detrimental effect of NEMO mutations in patients suffering from X-linked ectodermal dysplasia and immunodeficiency.
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http://dx.doi.org/10.1016/j.cell.2009.03.007DOI Listing
March 2009
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