Silmitasertib (CX-4945), a Clinically Used CK2-Kinase Inhibitor with Additional Effects on GSK3β and DYRK1A Kinases: A Structural Perspective

A clinical casein kinase 2 inhibitor, CX-4945 (silmitasertib), shows significant affinity toward the DYRK1A and GSK3β kinases, involved in down syndrome phenotypes, Alzheimer’s disease, circadian clock regulation, and diabetes. This off-target activity offers an opportunity for studying the effect of the DYRK1A/GSK3β kinase system in disease biology and possible line extension. Motivated by the dual inhibition of these kinases, we solved and analyzed the crystal structures of DYRK1A and GSK3β with CX-4945. We built a quantum-chemistry-based model to rationalize the compound affinity for CK2α, DYRK1A, and GSK3β kinases. Our calculations identified a key element for CK2α’s subnanomolar affinity to CX-4945. The methodology is expandable to other kinase selectivity modeling. We show that the inhibitor limits DYRK1A- and GSK3β-mediated cyclin D1 phosphorylation and reduces kinase-mediated NFAT signaling in the cell. Given the CX-4945’s clinical and pharmacological profile, this inhibitory activity makes it an interesting candidate with potential for application in additional disease areas

Nutlin-3a-aa: Improving the Bioactivity of a p53/MDM2 Interaction Inhibitor by Introducing a Solvent-Exposed Methylene Group

Nutlin-3a is a reversible inhibitor of the p53/MDM2 interaction. We have synthesized the derivative Nutlin-3a-aa bearing an additional exocyclic methylene group in the piperazinone moiety. Nutlin-3a-aa is more active than Nutlin-3a against purified wild-type MDM2, and is more effective at increasing p53 levels and releasing transcription of p53 target genes from MDM2-induced repression. X-ray analysis of wild-type MDM2-bound Nutlin-3a-aa indicated that the orientation of its modified piperazinone ring was altered in comparison to the piperazinone ring of MDM2-bound Nutlin-3a, with the exocyclic methylene group of Nutlin-3a-aa pointing away from the protein surface. Our data point to the introduction of exocyclic methylene groups as a useful approach by which to tailor the conformation of bioactive molecules for improved biological activity.This work was generously supported by the Deutsche Forschungsgemeinschaft (BE 4572/3-1 to T.B.). We extend our thanks to Barbara Klüver, Katrin Eckhardt, Nadiya Brovchenko, and Domenique Herbstritt for experimental support. Parts of the data described in this manuscript have been published in the dissertation of Florian Nietzold (Leipzig University, 2019).31 In addition, this work was financially supported by the National Science Centre, Poland (NCN) under Grant Symphony 2014/12/W/NZ1/00457 (to T.A.H). We thank HZB for the allocation of synchrotron radiation beamtime. We acknowledge the MCB Structural Biology Core Facility (supported by the TEAM TECH CORE FACILITY/2017-4/6 grant from the Foundation for Polish Science) for valuable support. Open Access funding enabled and organized by Projekt DEAL

Structural analysis of PIM1 kinase complexes with ATP-competitive inhibitors

Abstract PIM1 is an oncogenic kinase overexpressed in a number of cancers where it correlates with poor prognosis. Several studies demonstrated that inhibition of PIM1 activity is an attractive strategy in fighting overexpressing cancers, while distinct structural features of ATP binding pocket make PIM1 an inviting target for the design of selective inhibitors. To facilitate development of specific PIM1 inhibitors, in this study we report three crystal structures of ATP-competitive inhibitors at the ATP binding pocket of PIM1. Two of the reported structures (CX-4945 and Ro-3306) explain the off-target effect on PIM1 of respectively casein kinase 2 and cyclin-dependent kinase 1 dedicated inhibitors. In turn, the structure with CX-6258 demonstrates a binding mode of a potent, selective inhibitor of PIM1, PIM2, PIM3 and Flt-3 kinases. The consequences of our findings for future inhibitor development are discussed

Structure of the Complex of Human Programmed Death 1, PD-1, and Its Ligand PD-L1

SummaryTargeting the PD-1/PD-L1 immunologic checkpoint with monoclonal antibodies has recently provided breakthrough progress in the treatment of melanoma, non-small cell lung cancer, and other types of cancer. Small-molecule drugs interfering with this pathway are highly awaited, but their development is hindered by insufficient structural information. This study reveals the molecular details of the human PD-1/PD-L1 interaction based on an X-ray structure of the complex. First, it is shown that the ligand binding to human PD-1 is associated with significant plasticity within the receptor. Second, a detailed molecular map of the interaction surface is provided, allowing definition of the regions within both interacting partners that may likely be targeted by small molecules

HtrA trimers; PD-PDZ(1) in surface representation.

<p>L3 and the linker are shown as orange threads, L2, L1 and LD as blue threads. (<b>A)</b> wtHtrA2-peptide starting structure in orientation identical to that in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161526#pone.0161526.g003" target="_blank">Fig 3</a> bottom-right. (<b>B)</b> wtHtrA2-peptide after 50 ns MD. (<b>C</b>) active DegP PDB (entry 3CS0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161526#pone.0161526.ref017" target="_blank">17</a>]) PDZ2 is omitted for clarity. In contrast to <b>A</b> and <b>B</b>, where L3 protrudes outside on the convex side of the trimer, in the active DegP and in other active HtrAs (not shown) L3 enters between PD and PDZ, onto the trimer’s concave, contributing (vide bottom) to the allosteric activation cascade: L3*-LD-L1-L2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161526#pone.0161526.ref035" target="_blank">35</a>].</p

The restraints used in restrained MD of 1LCY monomer.

<p>The values in the middle column are inferences affixed to tendencies given in Fig 1 B,C in Ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161526#pone.0161526.ref038" target="_blank">38</a>].</p

HtrA2 trimers after 50 ns MD, with reference to the starting structures: The pink PDZ (ribbons) make 3 lids to the PD (surface) of the trimer viewed perpendicular to C3 as in Fig 3.

<p><b>Left:</b> The resultant <b>wtHtrA2-ligand</b> complex. PDZ domains, in Units A, B, C, (cyan, canary and amber ribbons, respectively) have moved equatorially (budding-flower-like) relative to their starting (pink) positons, especially in units A & C thus opening the PD-PDZ interface. <b>Right:</b> The resultant <b>HtrA2</b>^{<b>S306A/V325D</b>} <b>double mutant-ligand</b> complex. Moving PDZ domains are colored as in the left panel. Only minor PDZ motions are seen; no opening of the PD-PDZ takes place.</p

Overview of simulation results.

<p>For a viewer having the N-terminus and PD C-terminal barrel in the foreground, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161526#pone.0161526.g005" target="_blank">Fig 5</a>, “cc” and “c” denote, respectively, counterclockwise and clockwise rotation of PDZ versus PD. The viewer sees this rotation roughly round an axis parallel to α4 and passing for “cc” between α4 and α7, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161526#pone.0161526.g005" target="_blank">Fig 5</a>, and; for “c” by the peptide-binding motif ³⁶¹YIGV³⁶⁴. “Δ”applies to “cc” only and refers to another measure of extent of the “cc” rotation, viz. to its associated arc at maximized radius. I179(β2) and L398(β15) roughly fit this radius tips, hence their C^α-C^α (vs. ~6Ǻ at the start) distance increases with “cc” rotation defining “Δ“. “i” indicates minute irregular motions.</p

Human HtrA2 topology.

<p>(<b>A</b>) Protease domain: the N- and C-terminal helices, α1/α2 and α4, respectively, are omitted for clarity. The catalytic triad His, Asp, Ser and the loops are labeled according to the chymotrypsin nomenclature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161526#pone.0161526.ref016" target="_blank">16</a>]. (<b>B</b>) PDZ domain. The carboxylate binding loop is indicated.</p

Modeling statistics of the starting structures.

<p>Modeling statistics of the starting structures.</p