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Druggable Transient Pockets in Protein Kinases

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Druggable Transient Pockets in Protein Kinases molecules Review Druggable Transient Pockets in Protein Kinases Koji Umezawa 1 and Isao Kii 2,* 1 Department of Biomolecular Innovation, Institute for Biomedical Sciences, Shinshu University, 8304 Minami-Minowa, Kami-ina, Nagano 399-4598, Japan; [email protected] 2 Laboratory for Drug Target Research, Faculty & Graduate School of Agriculture, Shinshu University, 8304 Minami-Minowa, Kami-ina, Nagano 399-4598, Japan * Correspondence: [email protected]; Tel.: +81-265-77-1521 Abstract: Drug discovery using small molecule inhibitors is reaching a stalemate due to low se- lectivity, adverse off-target effects and inevitable failures in clinical trials. Conventional chemical screening methods may miss potent small molecules because of their use of simple but outdated kits composed of recombinant enzyme proteins. Non-canonical inhibitors targeting a hidden pocket in a protein have received considerable research attention. Kii and colleagues identified an inhibitor targeting a transient pocket in the kinase DYRK1A during its folding process and termed it FINDY. FINDY exhibits a unique inhibitory profile; that is, FINDY does not inhibit the fully folded form of DYRK1A, indicating that the FINDY-binding pocket is hidden in the folded form. This intriguing pocket opens during the folding process and then closes upon completion of folding. In this review, we discuss previously established kinase inhibitors and their inhibitory mechanisms in comparison with FINDY. We also compare the inhibitory mechanisms with the growing concept of “cryptic inhibitor-binding sites.” These sites are buried on the inhibitor-unbound surface but become apparent when the inhibitor is bound. In addition, an alternative method based on cell-free protein synthesis of protein kinases may allow the discovery of small molecules that occupy these mysterious binding sites. Transitional folding intermediates would become alternative targets in drug discovery, enabling the efficient development of potent kinase inhibitors. Citation: Umezawa, K.; Kii, I. Druggable Transient Pockets in Keywords: folding intermediate; cryptic binding site; protein kinase; thermodynamic equilibrium; Protein Kinases. Molecules 2021, 26, DYRK1A; FINDY; chemical screening 651. https://doi.org/10.3390/ molecules26030651 Academic Editor: Steven Verhelst 1. Protein Kinases Received: 23 December 2020 Enzymatic phosphorylation of a protein substrate with adenosine triphosphate (ATP) Accepted: 26 January 2021 was described in 1954 [1]. Thereafter, human genome sequencing revealed more than Published: 27 January 2021 500 protein kinases, which have been classified according to sequence homology-based clustering and designated the kinome [2,3]. Publisher’s Note: MDPI stays neutral Protein kinases are of therapeutic interest because aberrant kinase activity due to with regard to jurisdictional claims in genetic and sporadic mutation, chromosomal translocation, and/or gene amplification can published maps and institutional affil- cause diseases such as cancer, diabetes, inflammation, and neurodegeneration [4,5]. The iations. first clinical protein kinase inhibitor, fasudil (HA-1077), was approved in 1995 for cerebral vasospasm [6], and thereafter, many protein kinase inhibitors have been approved. The remarkable economic success of imatinib, approved in 2001 as a small molecule inhibitor of Abelson tyrosine kinase (ABL), which is dysregulated by chromosome translocation Copyright: © 2021 by the authors. in chronic myelogenous leukemia, kindled drug research and development targeting the Licensee MDPI, Basel, Switzerland. protein kinases involved in other cancers and diseases [7–9]. However, the U.S. Food and This article is an open access article Drug Administration–approved inhibitors only target approximately 20 protein kinases in distributed under the terms and the human kinome [4,8]. Thus, the human kinome is still an attractive drug target with a conditions of the Creative Commons significant research scope [4,10]. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Molecules 2021, 26, 651. https://doi.org/10.3390/molecules26030651 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 16 Molecules 2021, 26, 651 2 of 16 1.1. Structure of the Kinase Domain 1.1. Structure of the Kinase Domain The catalytic domain of protein kinases shares a conserved core consisting of two The catalytic domain of protein kinases shares a conserved core consisting of two lobes: lobes: the N-lobe, featuring a β-sheet with five strands, and the C-lobe, mostly comprising the N-lobe, featuring a β-sheet with five strands, and the C-lobe, mostly comprising α- α-helices and loops (Figure 1). A hinge region connects these lobes, forming an ATP-bind- helices and loops (Figure1). A hinge region connects these lobes, forming an ATP-binding ing cleft, a highly conserved catalytic scaffold [11,12], between these two lobes. ATP and cleft, a highly conserved catalytic scaffold [11,12], between these two lobes. ATP and the thesubstrate substrate bind bind to the to the cleft cleft and and a nearby a nearby recognition recognition site, site, respectively; respectively; the gamma the gamma phosphate phos- phateof ATP of isATP transferred is transferred onto a onto hydroxyl a hydroxyl group ofgroup the substrateof the substrate [13–16]; [13 the–16 resultant]; the resultant ADP is ADPreleased is released from the from cleft [the17– 19cleft]; and [17 ATP–19]; isand re-supplied ATP is re to-supplied the cleft to to drive the thecleft catalytic to drive cycle. the catalytic cycle. A B C N-lobe pY393 pY393 pY393 G383 Hinge P F382 D381 Activation loop E409 A407 C-lobe P408 FigureFigure 1. 1. StructureStructure of ofthe the kinase kinase domain. domain. (A) Ribbon (A) Ribbon representation representation of the ofkinase the kinase domain domain of ABL of (ProteinABL (Protein Data Bank Data (PDB) Bank ID: (PDB) 2V7A). ID: 2V7A).Blue and Blue orange and regions orange regionsindicate indicatethe N- and the C N--lobes, and C-lobes,respec- tively.respectively. Gray and Gray green and loops green represent loops represent the hinge the region hinge region and activation and activation loop, respectively. loop, respectively. The ty- The rosinetyrosine residue residue at position at position 393 393 in inthe the activation activation loop loop is isphosphorylated phosphorylated as as indicated. indicated. The The conserved conserved aminoamino acid acid residues residues (DFG (DFG and and APE APE motifs) motifs) in inand and around around the the activation activation loop loop are are also also indicated. indicated. (B) Molecular surface image of the ABL domain (PDB ID: 2V7A), in which the colors are identical (B) Molecular surface image of the ABL domain (PDB ID: 2V7A), in which the colors are identical to the regions shown in (A). ATP binds to the cleft between the N- and C-lobes. (C) Graphical rep- to the regions shown in (A). ATP binds to the cleft between the N- and C-lobes. (C) Graphical resentation of the kinase domain. representation of the kinase domain. ProteinProtein kinases alsoalso possess possess a flexiblea flexible and and non-structured non-structured element element (length: (length: 20–30 amino20–30 aminoacid residues) acid residues) in the C-lobe,in the C termed-lobe, termed the activation the activation loop. This loop. loop This begins loop begins with an with invari- an invariantant “DFG” “DFG” motif motif (Asp-Phe-Gly) (Asp-Phe-Gly) near near the ATP-binding the ATP-binding cleft cleft (Figure (Figure1)[ 20 1),21 [20,] and21] isand in is a intransition a transition from from disordered disordered to ordered to ordered upon upon activation. activation The. The carboxyl-terminal carboxyl-terminal regions regions of ofthis this loop loop are are designated designated the the “P+1 “P+1 loop” loop” and and the “APE”the “APE” motif motif (Ala-Pro-Glu). (Ala-Pro-Glu). The P+1The loopP+1 loopforms forms a pocket a pocket that bindsthat binds to residues to residues in the in substrate the substrate peptide peptide [22]. The[22]. conserved The conserved APE APEmotif motif stabilizes stabilizes the activationthe activation loop loop and theand P+1 the loop.P+1 loop. 1.2.1.2. Nascent Nascent Kinases Kinases ProteinProtein kinases kinases are are translated translated from from mRNA mRNA as as polypeptides, polypeptides, then then folding folding into into a a ther- ther- modynamicallymodynamically stable stable state state ( (FigureFigure 22).). NascentNascent kinaseskinases withoutwithout anyany post-translationalpost-translational modificationsmodifications generally generally take take an an inactive inactive conformation, conformation, or or latent conformation, in whichwhich ATPATP and and the substratesubstrate cannotcannot bind bind to to the the kinase kinase with with the the typical typical affinity. affinity. The activationThe activation loop loopnear near theATP-binding the ATP-binding cleft blockscleft blocks ATP andATP substrate and substrate binding. binding In the. In inactive the inactive conformation, confor- mation,the DFG the motif DFG ismotif typically is typically positioned positioned outside outside of the of active the active site site in the in the cleft, cleft, exposed exposed at atthe the surface surface of of the the kinase. kinase. Thus, Thus, inactiveinactive statesstates ofof kinaseskinases areare alsoalso denoteddenoted asas DFG-outDFG-out (Figure(Figure 3).3). Several studies have demonstrated that the N-lobe in the inactive kinase tends to be
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