Synthesis and Evaluation of Noncovalent Naphthalene-Based KEAP1-NRF2 Inhibitors.

pubs.acs.org/acsmedchemlett Letter Synthesis and Evaluation of Noncovalent Naphthalene-Based KEAP1-NRF2 Inhibitors Phillip R. Lazzara, Atul D. Jain, Amanda C. Maldonado, Benjamin Richardson, Kornelia J. Skowron, Brian P. David, Zamia Siddiqui, Kiira M. Ratia, and Terry W. Moore* Cite This: ACS Med. Chem. Lett. 2020, 11, 521−527 ACCESS Metrics & More Read Online sı Supporting Information * Article Recommendations ABSTRACT: The oxidative stress response, gated by the protein− protein interaction of KEAP1 and NRF2, has garnered significant interest in the past decade. Misregulation in this pathway has been implicated in disease states such as multiple sclerosis, rheumatoid arthritis, and diabetic chronic wounds. Many of the known activators of NRF2 are electrophilic in nature and may operate through several biological pathways rather than solely through the activation of the oxidative stress response. Recently, our lab has reported a nonelectrophilic, monoacidic, naphthalene-based NRF2 activator which exhibited good potency in vitro. Herein, we report a detailed structure−activity relationship of naphthalene-based NRF2 activators, an X-ray crystal structure of our monoacidic KEAP1 inhibitor, and identification of an underexplored area of the NRF2 binding pocket of KEAP1. KEYWORDS: KEAP1, NRF2, protein−protein interaction, oxidative stress C There have been multiple reports in recent years of nonelectrophilic KEAP1-NRF2 inhibitors with significant structural diversity, including various small molecules (1a− 1j) and peptides (1k) (Chart 1). Most of these molecules possess anionic character at physiological pH. Due to the relative ease of modifying compounds such as naphthalene 1a, we and others have developed an SAR of these compounds via scaffold-hopping approaches and modification to the flanking benzenesulfonamide arms; however, comparatively little investigation has been done to probe variations in the regions that link the naphthalene core to the benzensulfonamides.20,28 In this Letter, we present structural modifications, informed by a crystal structure of monoacid inhibitor 1c (Figure 2), that provide valuable insights into the key interactions governing the potency and binding affinities of these 1,4-disubstituted naphthalene inhibitors. Previously, we were unable to obtain a suitable cocrystal structure of 1c with the KEAP1 Kelch domain, so we analyzed the potential binding mode of monoacidic inhibitor 1c in silico.20 Docking experiments predicted that the carboxylate would likely interact with R483 and R415. We have now achieved success in cocrystallization of monoacidic inhibitor 1c hronic oxidative stress is implicated in a number of disease states, such as chronic obstructive pulmonary disease (COPD), multiple sclerosis, diabetic chronic wounds, and chronic kidney disease.1−6 Upregulating cellular defenses against oxidative stress may be a viable pathway for treatment or management of such diseases.7−9 NRF2 (nuclear factor (erythroid-derived 2)-like 2), a basic leucine zipper protein, regulates transcription of many antioxidant proteins. This oxidative stress response is gated primarily by the protein KEAP1 (Kelch-like ECH-associated protein 1), which sequesters NRF2 and, through a multiprotein assembly, polyubiquitinates it, marking it for proteosomal degradation.10 If the KEAP1-NRF2 protein−protein interaction is inhibited, NRF2 can no longer be sequestered and tagged for degradation. Inhibiting KEAP1 in this manner allows cytoplasmic NRF2 concentrations to increase, translocate into the nucleus, and promote the transcription of genes associated with the antioxidant response, such as NADPH quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO1), and glutamate cysteine ligases-C and -M (Figure 1).10−14 The KEAP1-NRF2 interaction is inhibited in the presence of electrophiles, reactive oxygen species, or reactive nitrogen species, leading to a cytoprotective response in the cell.15 Some therapies that inhibit the KEAP1-NRF2 interaction utilize KEAP1’s sensitivity to electrophiles to increase cellular NRF2 levels. Some electrophiles may be promiscuous binders, and their lack of selectivity may make identification of mechanism of action more challenging.16,17 © 2020 American Chemical Society Received: December 19, 2019 Accepted: February 19, 2020 Published: February 19, 2020 521 https://dx.doi.org/10.1021/acsmedchemlett.9b00631 ACS Med. Chem. Lett. 2020, 11, 521−527 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett Letter Figure 1. Top: KEAP1-NRF2 interaction under basal conditions. Bottom: Mechanism of NRF2 via electrophilic and nonelectrophilic pathways. Chart 1. Representative Examples of Known KEAP1 Inhibitors18−27 Our investigation into the structure−activity relationship began by probing the necessity of the bis-sulfonamide motif. In previous work, we showed that monoacidic analogs of 1b still maintained significant potency; however, no dicarboxylmonosulfonamide analogs were synthesized. We sought to determine if protein binding was driven more by the sulfonamide oxygens or the interactions of the carboxylates. A series of these monosulfonamide compounds were easily accessed through a Heck reaction of 1-bromo-4-aminonaphthalene (2) and subsequent derivatization (Scheme 1). Although these monosulfonamide compounds are structurally similar to reported monosulfonamide compound RA-839 (1f), none of these compounds retained nanomolar affinities for the Kelch-domain of KEAP1, as determined by fluorescence anisotropy (FA).21,29 This difference in affinity may be due to the rigidity of the acid in RA-839 versus the more freely rotating carboxylate groups in 3 and 4. Even though compounds 5 and 6 contained an electrophilic α,β-unsaturated carbonyl, the activity of these compounds in the FA assay would be solely dependent on nonelectrophilic inhibition since with the Kelch domain of KEAP1 from a sodium formate solution. The cocrystal structure that we obtained contained a unit cell comprised of four Kelch domains, each possessing 1c in slightly different orientations. Two Kelch domains contained a formate ion interacting with the unsubstituted sulfonamide, while the remaining two displayed water molecules in this position. While these two variations contained slightly different orientations, the overall interactions between 1c and the Kelch domain remained similar (Figure 2A and Figure 2B). Interestingly, we observed that the carboxymethyl functionality is engaged in a hydrogen bond network and dipolar interactions with R415, N414, N382, S363, and a water molecule, which was contradictory with our docking experiments, which showed interactions with R415 and R483.20 In the crystal structure, key interactions appear to be made between the sulfonamide oxygen atoms and S363, S508, Y525, S555, S602 and two water molecules. With these data in hand, we set forth to determine which of these interactions are critical for inhibitor binding. 522 https://dx.doi.org/ (...truncated)