X‐ray Structures and Feasibility Assessment of CLK2 Inhibitors for Phelan–McDermid Syndrome

Abstract

CLK2 inhibition has been proposed as a potential mechanism to improve autism and neuronal functions in Phelan-McDermid syndrome (PMDS). Herein, the discovery of a very potent indazole CLK inhibitor series and the CLK2 X-ray structure of the most potent analogue are reported. This new indazole series was identified through a biochemical CLK2 Caliper assay screen with 30k compounds selected by an in silico approach. Novel high-resolution X-ray structures of all CLKs, including the first CLK4 X-ray structure, bound to known CLK2 inhibitor tool compounds (e.g., TG003, CX-4945), are also shown and yield insight into inhibitor selectivity in the CLK family. The efficacy of the new CLK2 inhibitors from the indazole series was demonstrated in the mouse brain slice assay, and potential safety concerns were investigated. Genotoxicity findings in the human lymphocyte micronucleus test (MNT) assay are shown by using two structurally different CLK inhibitors to reveal a major concern for pan-CLK inhibition in PMDS.

Cdc-Like Kinases (CLKs): Biology, Chemical Probes, and Therapeutic Potential

“…Table 2 contains the published values obtained in primary biochemical assays; however, for their direct comparison it is important to keep in mind that these data were often obtained from various sources/assays or from the same (similar) assay but under unequal conditions (e.g., using different concentrations of ATP). On the other hand, some studies report a direct comparison of several (usually two or three) inhibitors from different classes/publications, profiling them in the same assay [ 76 , 133 , 134 , 135 , 136 ].…”

“…CX-4945 ( Figure 10 ) has been primarily reported, marketed, and used as a highly selective and potent inhibitor of CK2 since 2011 [ 140 ]. However, CX-4945 strongly inhibits also CLKs and DYRK1A [ 133 ]. In addition, it interacts significantly with other kinases such as HIPKs or PIM kinases [ 133 , 139 ].…”

Emerging roles of the αC‐β4 loop in protein kinase structure, function, evolution, and disease

“…Protein names are provided alongside its kinase group. Locations for αC‐β4 loops are provided: 93–135 in scCKA1 (PDB ID: 4jr7), 147–160 in SRPK2 (PDB ID: 2x7g), 253–262 in HIPK2 (PDB ID: 6p5s), 212–222 in CLK3 (PDB ID: 6fyr), 92–112 in VRK1 (PDB ID: 6bru), 95–106 in Gilgamesh (PDB ID: 4 nt4), and 322–340 in Rhoptry kinase (PDB ID: 3byv) . More information about these structures can be found in Table .…”

The crystal structure of the protein kinase HIPK2 reveals a unique architecture of its CMGC-insert region

“…We tested a panel of inhibitors known to act on HIPK2 and a broad range of CMGC family kinases (58 -60). Under the conditions tested, HIPK2 crystallized in the presence of the CK2␣ ATP-competitive inhibitor CX-4945, previously shown to inhibit HIPK2, HIPK3, and a number of other kinases (62,76), and crystallized in complex with CK2␣, the proviral integration site for Moloney murine leukemia virus-1 kinase (Pim1), and CLK2-4 kinases (63)(64)(65).…”

“…In HIPK2, the ATP-pocket is formed by Leu 205 , Val 213 , Ala 226 , Val 261 , Phe 277 , Met 279 , Leu 280 , Met 331 , and Ile 345 . The naphthyridine ring of CX-4945 is tightly bound by the R-spine residues Previous crystal structures have determined the mode of CX-4945 binding to CK2␣ (63,76), Pim1 (64), and CLK2-4 (65). The core hydrophobic active-site residues are well-conserved between HIPK2, CK2␣, and the CLKs, whereas the sequence of the Pim1 hinge region is different at several posi- EDITORS' PICK: Structure of HIPK2 bound to CX-4945 tions, which make contact with CX-4945 ( Fig.…”