Berberine Directly Targets the NEK7 Protein to Block the NEK7–NLRP3 Interaction and Exert Anti-inflammatory Activity

Abstract

Berberine (BBR), a traditional Chinese medicine, has therapeutic effects on a variety of inflammation-related diseases, but its direct proteomic targets remain unknown. Using activity-based protein profiling, we first demonstrated that BBR directly targets the NEK7 protein via the hydrogen bond between the 2,3-methylenedioxy and 121-arginine (R121) residues. The fact that R121 is located precisely within the key domain involved in the NEK7–NLRP3 interaction allows BBR to specifically block the NEK7–NLRP3 interaction and successively inhibit IL-1β release, independent of the NF-κB and TLR4 signaling pathways. Moreover, BBR displays in vivo anti-inflammatory efficacy in a NEK7-dependent manner. Therefore, we consider NEK7 to be a key target of BBR in the treatment of NLRP3-related inflammatory diseases, and the development of novel NEK7–NLRP3 interaction inhibitors might be easily achieved using NEK7 as a target.

Capture and Identification of Dual Specificity Mitogen-Activated Protein Kinase Kinase 7 as a Direct Proteomic Target of Berberine to Affect the c-JunN-Terminal Kinase Pathway

“…Our previous studies demonstrate that BBR directly targets the NEK7 protein and specifically blocks the NEK7-NLRP3 interaction and successively inhibits IL-1β release and its related inflammatory diseases. 24 MAP2K7 is another direct target of BBR to exert various effects related to the JNK pathway. As depicted in Figure 7, these results enable us to further understand the BBR's multi-target synergy effects inflammation-Page 14 of 21 CCS Chemistry related diseases at the intersection of JNK and NLRP3 pathways, such as obesity and insulin resistance.…”

“…1 Over the past 20 years, its multiple pharmacological activities have been discovered and elucidated, including antiinflammatory, antitumor, hypoglycemic, hypolipidemic, antivirus, etc, [2][3][4][5][6][7][8][9][10][11] suggesting a typical multitargetdirected mechanism of BBR. A couple of classical cellular pathways, such as NIMA-related kinase 7 (NEK7)/nucleotide-binding domain-like receptor protein 3 (NLRP3) and c-Jun N-terminal protein kinases (JNK)/stress activated protein kinases (SAPKs), as well as interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STATs), 8,[12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] have been proven to closely account for BBR's polypharmacological effects. Furthermore, several direct proteomic targets of BBR, such as NEK7, UHRF1, RXRα, actin and ephrin-B2, 18,24,25,28,29 have been identified to explain its synergistic effects.…”

“…A couple of classical cellular pathways, such as NIMA-related kinase 7 (NEK7)/nucleotide-binding domain-like receptor protein 3 (NLRP3) and c-Jun N-terminal protein kinases (JNK)/stress activated protein kinases (SAPKs), as well as interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STATs), 8,[12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] have been proven to closely account for BBR's polypharmacological effects. Furthermore, several direct proteomic targets of BBR, such as NEK7, UHRF1, RXRα, actin and ephrin-B2, 18,24,25,28,29 have been identified to explain its synergistic effects. 24,30,31 The JNK pathway is mainly activated by various damage associated molecular patterns (DAMPs) including proinflammatory cytokines (tumor necrosis factor (TNF)-α and IL-1α), and the stimulation of JNK pathway gives rise to a marked increase of JNK phosphorylation (p-JNK) level, which lead to the progression of inflammatory, neurodegenerative and cell proliferative related diseases.…”

“…Furthermore, several direct proteomic targets of BBR, such as NEK7, UHRF1, RXRα, actin and ephrin-B2, 18,24,25,28,29 have been identified to explain its synergistic effects. 24,30,31 The JNK pathway is mainly activated by various damage associated molecular patterns (DAMPs) including proinflammatory cytokines (tumor necrosis factor (TNF)-α and IL-1α), and the stimulation of JNK pathway gives rise to a marked increase of JNK phosphorylation (p-JNK) level, which lead to the progression of inflammatory, neurodegenerative and cell proliferative related diseases. 12,15,25,26 It is worth noting that BBR could suppress p-JNK to alleviate inflammation-related symptoms, such as obesity, insulin resistance, and exert effects on tumorous and neurodegenerative diseases.…”

“…Then, taking the active probe as a chemical tool, the direct proteomic target of BBR upon the JNK pathway was captured and identified using activitybased protein profiling (ABPP), the cellular thermal shift assay (CETSA) as well as other chemical proteomics techniques (Figure 1) to clarify how BBR affects the JNK pathway. As depicted in Scheme 2, taking compound 8 24 with a 10-hydroxyl as the starting material, photoaffinity…”