Second-Generation AUTACs for Targeted Autophagic Degradation.

Targeted protein degradation via the ubiquitin-proteasome system has emerged as one of the most promising drug discovery modalities. Autophagy, another intracellular degradation system, can target a wide range of nonproteinous substrates as well as proteins, but its application to targeted degradation is still in its infancy. Our previous work revealed a relationship between guanine modification of cysteine residues on intracellular proteins and selective autophagy, resulting in the first autophagy-based degraders, autophagy-targeted chimeras (AUTACs). Based on the research background, all the reported AUTACs compounds contain cysteine as a substructure. Here, we examine the importance of this substructure by conducting SAR studies and report significant improvements in the degrader's activity by replacing cysteine with other moieties. Several derivatives showed sub-µM range degrading activity, demonstrating the increased practical value of AUTACs.

Discovery of a novel series of indolinylpyrimidine-based GPR119 agonists: Elimination of ether-a-go-go-related gene liability using a hydrogen bond acceptor-focused approach.

We previously identified a novel series of indolinylpyrimidine derivatives exemplified by 2 in Figure 1, which is an indoline based derivative, as potent GPR119 agonists. Despite the attractive potency of 2, this compound inhibited the human ether-a-go-go-related gene (hERG) K+ channel. We elucidated crucial roles of the methylsulfonyl group of 2 in its interaction with the hERG channel and the GPR119 receptor, presumably as a hydrogen bond acceptor (HBA). To remove the undesirable hERG inhibitory activity, a strategy was implemented to arrange an HBA on a less conformationally flexible framework at the indoline 5-position instead of the methylsulfonyl group. This successfully led to the discovery of a piperidinone ring as a desirable motif at the indoline 5-position, which could minimize hERG liability as shown by 24b. Further optimization focused on the reduction of lipophilicity in terms of more favorable drug-like properties. Consequently, the introduction of a hydroxy group at the 3-position of the piperidinone ring effectively reduced lipophilicity without compromising GPR119 potency, resulting in the identification of (3S)-3-hydroxy-1-{1-[6-({1-[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-yl}oxy)pyrimidin-4-yl]- 2,3-dihydro-1H-indol-5-yl}piperidin-2-one ((S)-29) as a novel, potent, and orally bioavailable GPR119 agonist with a well-balanced profile. The pharmacological effects of this compound were also confirmed after single and chronic oral administration in diabetic animal models.

Discovery of Novel 5-(Piperazine-1-carbonyl)pyridin-2(1H)-one Derivatives as Orally eIF4A3-Selective Inhibitors.

Starting from our previous eIF4A3-selective inhibitor 1a, a novel series of (piperazine-1-carbonyl)pyridin-2(1H)-one derivatives was designed, synthesized, and evaluated for identification of orally bioavailable probe molecules. Compounds 1o and 1q showed improved physicochemical and ADMET profiles, while maintaining potent and subtype-selective eIF4A3 inhibitory potency. In accord with their promising PK profiles and results from initial in vivo PD studies, compounds 1o and 1q showed antitumor efficacy with T/C values of 54% and 29%, respectively, without severe body weight loss. Thus, our novel series of compounds represents promising probe molecules for the in vivo pharmacological study of selective eIF4A3 inhibition.

Crystal Structure of a Human K-Ras G12D Mutant in Complex with GDP and the Cyclic Inhibitory Peptide KRpep-2d.

The Ras proteins play roles in cell differentiation, proliferation, and survival. Aberrant signaling through Ras-mediated pathways in tumor cells occurs as a result of several types of mutational damage, which most frequently affects the amino acids G12, G13, and Q61. Recently, KRpep-2d was identified as a K-Ras(G12D) selective inhibitory peptide against the G12D mutant of K-Ras, which is a key member of the Ras protein family and an attractive cancer therapeutic target. In this study, the crystal structure of the human K-Ras(G12D) mutant was determined in complex with GDP and KRpep-2d at 1.25 Å resolution. This structure revealed that the peptide binds near Switch II and allosterically blocks protein-protein interactions with the guanine nucleotide exchange factor. This discovery of a unique binding pocket provides valuable information that will facilitate the design of direct Ras inhibitors.

Investigation of the structural requirements of K-Ras(G12D) selective inhibitory peptide KRpep-2d using alanine scans and cysteine bridging.

A structure-activity relationship study of a K-Ras(G12D) selective inhibitory cyclic peptide, KRpep-2d was performed. Alanine scanning of KRpep-2d focusing on the cyclic moiety showed that Leu7, Ile9, and Asp12 are the key elements for K-Ras(G12D) selective inhibition of KRpep-2d. The cysteine bridging was also examined to identify the stable analog of KRpep-2d under reductive conditions. As a result, the KRpep-2d analog (12) including mono-methylene bridging showed potent K-Ras(G12D) selective inhibition in both the presence and the absence of dithiothreitol. This means that mono-methylene bridging is an effective strategy to obtain a reduction-resistance analog of parent disulfide cyclic peptides. Peptide 12 inhibited proliferation of K-Ras(G12D)-driven cancer cells significantly. These results gave valuable information for further optimization of KRpep-2d to provide novel anti-cancer drug candidates targeting the K-Ras(G12D) mutant.

K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology.

Amino-acid mutations of Gly12 (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH2) as a consensus sequence. KRpep-2 showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. KD and IC50 values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH2) that inhibited enzyme activity of K-Ras(G12D) with IC50 = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 µM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs.

Synthesis, structure-activity relationship, and pharmacological studies of novel melanin-concentrating hormone receptor 1 antagonists 3-aminomethylquinolines: reducing human ether-a-go-go-related gene (hERG) associated liabilities.

Recently, we discovered 3-aminomethylquinoline derivative 1, a selective, highly potent, centrally acting, and orally bioavailable human MCH receptor 1 (hMCHR1) antagonist, that inhibited food intake in F344 rats with diet-induced obesity (DIO). Subsequent investigation of 1 was discontinued because 1 showed potent hERG K(+) channel inhibition in a patch-clamp study. To decrease hERG K(+) channel inhibition, experiments with ligand-based drug designs based on 1 and a docking study were conducted. Replacement of the terminal p-fluorophenyl group with a cyclopropylmethoxy group, methyl group introduction on the benzylic carbon at the 3-position of the quinoline core, and employment of a [2-(acetylamino)ethyl]amino group as the amine portion eliminated hERG K(+) channel inhibitory activity in a patch-clamp study, leading to the discovery of N-{3-[(1R)-1-{[2-(acetylamino)ethyl]amino}ethyl]-8-methylquinolin-7-yl}-4-(cyclopropylmethoxy)benzamide (R)-10h. The compound (R)-10h showed potent inhibitory activity against hMCHR1 and dose-dependently suppressed food intake in a 2-day study on DIO-F344 rats. Furthermore, practical chiral synthesis of (R)-10h was performed to determine the molecule's absolute configuration.