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Computational Identification and Validation of Small-Molecule Inhibitors of NLRP3 Inflammasome through Biomolecular Simulations and Experimental Studies

초록/요약

The nucleotide-binding oligomerization domain (NOD)-like receptor family, pyrin domain-containing protein 3 (NLRP3) inflammasome is activated in response to a variety of stimuli. Pathogen-associated molecular patterns (PAMPs) and Damage-associated molecular patterns (DAMPs) derived by a range of pathogens, pore-forming toxins like nigericin, adenosine triphosphate, aggregates, and particulate crystals like uric acid crystals, all activate the NLRP3 inflammasome. The aberrant activation of NLRP3 inflammasome plays an essential role in complex diseases such as type 2 diabetes, multiple sclerosis, cryopyrin-associated periodic syndrome (CAPS), traumatic brain injury (TBI), psoriasis, and Alzheimer’s disease (AD). To combat with the NLRP3 inflammasome associated diseases, it is important to block the abnormal signaling of NLRP3. Therefore, two potential sites of NLRP3 were selected for the drug development. The first site (Site I) corresponding to hel-ical domain 2 (HD2), and leucine-rich repeat (LRR) of NLRP3 and overlapping with the NIMA-related kinase 7 (NEK7) binding site. The second site (Site II) cor-responding to the NACHT domain of NLRP3 where adenosine-5'-diphosphate (ADP) binds. A large multiconformational chemical library was utilized for the in silico virtual screening and molecular docking independently on these two sites. After close visual evaluation, several factor were considered such as interactions, chemical scaffold, binding score, and stability in the pocket. The 19 top-scoring consensus ligands designated as, NLRP3 inhibitory compounds (NIC1−NIC19) were experimentally validated to assess their inhibitory potential on the NLRP3-signaling pathway. The cell-based screening process led us to shortlist the NIC7 compound based on its better activity to inhibit the NLRP3- mediated release of IL-1β cytokine. To improve the activity, multiple derivatives were developed by modifying the main scaffold of NIC7. The in vitro assays confirmed that NIC7w is a better version of NIC7 to inhibit the activation of caspase 1 along with the secretion of interleukin (IL)-1β, IL-18, and lactate dehydrogenase (LDH). Nonetheless, western blot anal-ysis revealed the inhibition of key components of NLRP3 signaling pathways i.e. IL-1β and caspase-1. Furthermore, we also confirmed metabolic stability, and cy-tochrome P450 inhibition for NIC7, and NIC7w, which showed their better half-life and stability. In addition, molecular dynamics (MD) simulations were per-formed to obtain a comprehensive idea about the stability of both complexes NLRP3−NIC7 and NLRP3−NIC7w as a function of simulation time. We examined therapeutic potential of NIC7 in the diseased model of AD by ana-lyzing its effect on cognitive impairment as well as expression of dopamine recep-tors and neuronal marker. NIC7 significantly reversed the associated disease symp-toms into the mice model. On the other hand, NIC7 did not reverse the disease symptoms into the imiquimod (IMQ)-induced diseased model of psoriasis. This could be because of the development of the psoriasis model with IMQ but not with the IL-23, which involves the P2X7 receptor-mediated NLRP3-signaling pathway. It indicates that IMQ-based psoriasis is independent of NLRP3. Collectively, our results are consistent with previous findings suggesting that the model of IMQ-induced psoriasis does not mainly involve NLRP3, which hinders the reversal of disease conditions with our NLRP3- specific inhibitor, NIC7. Conclusively, NIC7 is a promising inhibitor of NLRP3-mediated signaling that can successfully alleviate the cognitive impairment observed during AD and, therefore, can act as a lead candidate for the development of NLRP3-specific therapeutics to treat AD or NLRP3-mediated diseases.

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목차

1. Introduction 1
1.1. Overview of Inflammasome 2
1.2. NLRP3 inflammasome 3
1.3. Mechanism of NLRP3 inflammasome activation 4
1.4. Disease prospects of NLRP3 6
1.4.1. NLRP3 role in Alzheimer's disease 6
1.4.2. NLRP3 role in psoriasis 7
1.5. Drug modulator of NLRP3 inflammasome 8
2. Methods 9
2.1. Computational section 10
2.1.1. Preparation of a virtual screening library 10
2.1.2 Analysis of molecular-fingerprint-based structure similarity 10
2.1.3. In silico protein structure preparation 11
2.1.4. Pharmacophore screening 11
2.1.5. The structure-based virtual screening 11
2.1.6. Identification of potent derivatives 12
2.1.7. MD simulations of NLRP3-NIC7 and NLRP3-NIC7w 13
2.2. Experimental section 13
2.2.1. Cell lines and reagents 13
2.2.2. A cell viability assay 14
2.2.3. Analysis of cytokines and lactate dehydrogenase (LDH) 14
2.2.4. Western blot analysis 15
2.2.5. Quantitative polymerase chain reaction (qPCR) 15
2.2.6. Immunohistochemistry 16
2.2.7. A microsomal stability assay 16
2.2.8. Cytochrome P450 inhibition 17
2.2.9. LC-MS/MS analysis 17
2.2.10. In vivo experiments in AD mouse model 18
2.2.11. Behavioral analysis 19
2.2.12. In vivo experiments in IMQ-induced psoriasis mouse model 19
2.2.13. Statistical analysis 20
3. Results and Discussion 21
3.1. Summary 22
3.2. Results 23
3.2.1. Identification of NIC7 as a potential lead against NLRP3 signaling 23
3.2.2. NIC7w shows greater potency than NIC7 26
3.2.3. NIC7 prevents memory impairment in the AD model 27
3.2.4. The lack of a benefit of NIC7 in IMQ-induced psoriasis mouse model 31
3.2.5. Stability and binding mode of NIC7 and NIC7w 32
3.2.6. Evaluating microsomal stability of NIC7 and NIC7w 34
3.3. Discussion 37
3.4. Conclusion 40
4. References 42

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