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.
The diazo ketones derived from Z‐ and Boc‐protected alanine, valine, and alanyl‐alanine (1–5) were decomposed by catalytic amounts of silver benzoate/Et3N in the presence of polyfunctional nucleophiles such as 3‐methyl‐1,3‐butanediol, 2‐aminoethanol, carbohydrates (xylofuranose and sucrose) and nucleosides (thymidine, adenosine derivatives). In many cases, products of β‐amino acylations are formed (6–14, 15b, 17–19, 21, 23, 24) with surprising functional group selectivities. The scope and limitations of the method are described.
Photolysis and Ag-benzoate-catalyzed decomposition of the diazo ketones 2 and 4 derived from Z-Ala-OH and Z-Ala-Ala-OH in the presence of oligonucleotide derivatives bearing at the 5'-terminus an NH, instead of the OH group, or an arninohexyl phosphate group lead to Z-protected 3-aminobutanoyl and to Z-Ala-b-HAla derivatives, respectively (conjugates 12, 13, and 17-23, Schemes 3-5). In solution, this amide-forming acylation reaction could be realized only with oligomers containing up to 8 unprotected nucleotide building blocks (Schemes 3 and 4). With the analogous polymer-bound and protected oligonucleotide derivatives as amino nucleophiles, excellent yields were obtained with all chain lengths tested (up to ISrner, Scheme 5 ) . The products were purified by reversed-phase HPLC and characterized by MALDI-TOF mass spectrometry (Figs. 2 4 , Table 2) and by capillary gel electrophoresis (Fig. 2). 1. Introduction. -Owing to the potential of oligonucleotides or peptides as therapeutic agents and the tremendous efforts put into the chemical development of these biopolymers over the past 25 years, conjugates such as those derived from oligonucleotides conjugated with peptides (also called oligonucleo-peptides) are attracting increasing interest. These conjugates offer a new chemical way to broaden the spectrum of properties of synthetic oligonucleotides [ 11. For example, conjugation of either cationic peptides such as PO~Y-L-LYS or poly-L-Arg or of hydrophobic groups such as tryptophan with an oligonucleotide can increase its cellular uptake [2]. Oligonucleotides have also been conjugated to peptide signal sequences [3] and a-helical peptides [4]. Recently, nucleopeptides were proposed as potential artificial nucleases [5] [6]. Furthermore, a lysine-rich peptide coupled to a DNA sequence was reported to show a 48 000 fold acceleration over its unmodified counterpart during duplex formation [7]. The combination of know-how acquired in both fields of peptide and oligonucleotide synthesis has triggered different synthetic strategies. Linear solid-phase syntheses of these hybrid molecules are described which required special protecting schemes due to chemical incompatibilities of standard oligonucleotide or peptide reactivities and properties [8]. 5'-Peptide-oligonucleotide con-
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