Structural modification of C2-substituents on 1,4-bis(arylsulfonamido)benzene or naphthalene-N,N'-diacetic acid derivatives as potent inhibitors of the Keap1-Nrf2 protein-protein interaction.

The Keap1-Nrf2-ARE signaling pathway is an attractive therapeutic target for the prevention and treatment of oxidative stress-associated diseases by activating the cellular expression of cytoprotective enzymes and proteins. Small molecule inhibitors can directly disrupt the Keap1-Nrf2 protein-protein interaction (PPI), resulting in elevated levels of Nrf2 protein and subsequent stimulation of related antioxidant responses. Previously, we found that 1,4-bis(arylsulfonamido)benzene or naphthalene-N,N'-diacetic acid derivatives with an ether type C2-substituent on the benzene or naphthalene core exhibited potent inhibitory activities with IC50's in the submicromolar or nanomolar range. We here describe a more detailed structure-activity relationship study around the C2 substituents containing various polar linkers shedding new insight on their binding interactions with the Keap1 Kelch domain. The key observation from our findings is that the substituents at the C2-position of the benzene or naphthalene scaffold impact their inhibitory potencies in biochemical assays as well as activities in cell culture. The biochemical FP and TR-FRET assays revealed that the naphthalene derivatives 17b and 18 with an additional carboxylate at the C2 were the most active inhibitors against Keap1-Nrf2 PPI. In the cell-based assay, the two compounds were shown to be potent Nrf2 activators of the transcription of the Nrf2-dependent genes, such as HMOX2, GSTM3, and NQO1.

Structure-activity relationships of 1,4-bis(arylsulfonamido)-benzene or naphthalene-N,N'-diacetic acids with varying C2-substituents as inhibitors of Keap1-Nrf2 protein-protein interaction.

The Keap1-Nrf2-ARE pathway plays an important role in responding to oxidative stress and maintaining the redox homeostasis. Small molecule inhibitors targeting directly the Keap1-Nrf2 protein-protein interaction (PPI) can potentially be developed into effective preventive and therapeutic agents for numerous chronic inflammatory diseases. To improve the drug-like properties and inhibitory potency of these inhibitors, a series of 1,4-bis(arylsulfonamido)benzene or naphthalene-N,N'-diacetic acids with varying substituents at C-2 position of the benzene or naphthalene core were designed and synthesized. Among them, compound 12d with 2-(4-fluorobenzyloxy) group was the most potent direct inhibitor of Keap1-Nrf2 PPI with an IC50 of 64.5 nM in the fluorescent polarization (FP) assay and 14.2 nM in a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Moreover, cell-based biological assay showed that 12d significantly increased the mRNA levels of Nrf2 downstream genes, GSTM3, HMOX2 and NQO1, through Nrf2 activation. The discovery of the new scaffolds possessing diverse O-linked fragments at the C2 position offers opportunities to further modify the chemical structures of Keap1-Nrf2 PPI inhibitors to improve their pharmacokinetic, efficacy and safety profiles.

Discovery of disubstituted xylylene derivatives as small molecule direct inhibitors of Keap1-Nrf2 protein-protein interaction.

The Keap1-Nrf2-ARE system represents a crucial antioxidant defense mechanism that protects cells against reactive oxygen species. Targeting Keap1-Nrf2 protein-protein interaction (PPI) has become a promising drug target for several oxidative stress-related and inflammatory diseases including pulmonary fibrosis, chronic obstructive pulmonary disorder (COPD) and cancer chemoprevention. For the development of a potential therapeutic agent, drug-like properties and potency are important considerations. In this work, we focused on the modification of 4 as a lead through a molecular dissection strategy in an effort to improve its metabolic stability, leading to the discovery of a series of new disubstituted xylylene derivatives. The preliminary SAR of 9a indicated that compound 21a containing S-methylated acetate moieties exhibited comparable potency to the lead compound 4 in a fluorescent polarization assay but with improved metabolic stability in the presence of human liver microsomes.

Discovery of direct inhibitors of Keap1-Nrf2 protein-protein interaction as potential therapeutic and preventive agents.

The Keap1-Nrf2-ARE pathway is an important antioxidant defense mechanism that protects cells from oxidative stress and the Keap1-Nrf2 protein-protein interaction (PPI) has become an important drug target to upregulate the expression of ARE-controlled cytoprotective oxidative stress response enzymes in the development of therapeutic and preventive agents for a number of diseases and conditions. However, most known Nrf2 activators/ARE inducers are indirect inhibitors of Keap1-Nrf2 PPI and they are electrophilic species that act by modifying the sulfhydryl groups of Keap1׳s cysteine residues. The electrophilicity of these indirect inhibitors may cause "off-target" side effects by reacting with cysteine residues of other important cellular proteins. Efforts have recently been focused on the development of direct inhibitors of Keap1-Nrf2 PPI. This article reviews these recent research efforts including the development of high throughput screening assays, the discovery of peptide and small molecule direct inhibitors, and the biophysical characterization of the binding of these inhibitors to the target Keap1 Kelch domain protein. These non-covalent direct inhibitors of Keap1-Nrf2 PPI could potentially be developed into effective therapeutic or preventive agents for a variety of diseases and conditions.