MOLECULAR ANALYSIS OF CHEMOKINE CCL2-INHIBITOR INTERACTIONS FOR REGULATING MONOCYTE TRAFFICKING

Abstract

The conventional representation of the innate immune response in epidemiological circumstances and inflammation entails the orchestrated activation and mobilization of diverse immune cell types to the sites of inflammation. The elimination of invading pathogens, harmful conditions, and other pathophysiological challenges is accomplished through the targeted recruitment of leukocytes to the inflamed locale, a phenomenon commonly referred to as leukocyte trafficking. Chemokines, commonly recognized as chemotactic cytokines, serve as pivotal factors in the orchestration of leukocyte migration. They govern and facilitate the migratory patterns and spatial distribution of leukocytes. The pivotal roles of chemokines encompass a diverse range of dynamic processes, spanning from the orchestration of immune cell localization in various disease states to the establishment and maintenance of immune system equilibrium. Chemokines represent the largest cytokine family, comprising over 50 members in both humans and mice. These are highly conserved, soluble chemotactic proteins with a molecular weight ranging from 8 to 10 kDa. Based on the arrangement of the initial two out of four cysteine residues, chemokines are classified into four distinct sub-families: C, CC, CXC, and CX3C chemokines. Chemokines are recognized for their ability to modulate various biological processes by engaging with their respective receptors (G-protein-coupled receptors, GPCRs) and cell surface glycosaminoglycans (GAGs). In the context of human biology, approximately 50 chemokines and roughly 20 chemokine receptors have been identified, thus establishing a multifaceted chemokine-receptor interplay. CC chemokines, also known as β-chemokines, are characterized by the presence of two consecutive cysteine amino acids at their N-terminal region, with disulfide bridges formed between the first and third cysteine and the second and fourth cysteine residues. Within the realm of mammals, approximately 28 different ligands/members of CC chemokines, denoted as CCL1 to CCL28, have been documented. Typically, most members of the CC chemokine family are distinguished by the presence of four cysteine residues and are referred to as C4-CC chemokines. Nonetheless, some members exhibit six cysteines, earning them the designation of C6-CC chemokines. The fundamental structural composition of CC chemokines encompasses an extended N-terminal section, followed by three antiparallel β-sheets and a Cterminal α-helix. These chemotactic proteins primarily induce the activation and migration of monocytes/macrophages. Based on their chromosomal positioning, two distinct gene groups have been identified: monocyte chemotactic proteins (MCPs) and macrophage inflammatory proteins (MIPs). The MCP sub-family of CC chemokines encompasses a cluster of four members, designated as MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, and MCP-4/CCL7. Among these four members, MCP-1, also widely recognized as CCL2, holds the distinction of being the first-discovered human CC chemokine and is considered the most pivotal member within the MCP family. CCL2 consists of 76 amino acids, resulting in an approximate molecular weight of 9 kDa. CCL2 has been linked to numerous detrimental conditions, including cardiovascular diseases, atherosclerosis, multiple sclerosis, cancerous conditions, and HIV infections. The activity of CCL2-CCR2-glycosaminoglycan network in GAG-induced oligomerization, monocyte migration and activation of multiple downstream signalling pathways has widely been acknowledged as a crucial process for the inflammatory responses against the abovementioned diseases. Natural products, especially the small molecules originating primarily from microorganisms and plants, have evolved over millions of years to regulate/modulate the diverse chemical signalling within biological systems. A substantial number of these molecules and their structural analogues have demonstrated exceptional efficacy as pharmaceutical agents, drug candidates, and tools for manipulating various processes in the biomedical realm, particularly in the treatment of infectious diseases and cancer. Among these natural products, flavonoids represent a significant class of plant-derived secondary metabolites. Due to their remarkable biological activities, including antioxidant, anti-inflammatory, anti-cancer, and antimicrobial properties, they have garnered attention as promising candidates in alternative medicine. Moreover, flavonoids are recognized as important constituents of the human diet. These activities of the flavonoids are outcomes of their interaction with the biologically significant macromolecules such as proteins, DNA, RNA, and membrane lipids. The detailed mechanistic insights of the underlying intricacies of such interactions yield valuable lead to the development of novel nutraceutical formulations. While a substantial body of literature has explored the molecular interactions between flavonoids and various proteins, their biophysical and atomistic interactions with immunomodulatory proteins such as chemokines yet remains sparsely explored. The present thesis delves into the structural dynamics of CCL2 chemokine. Both the wild-type and monomeric (P8A) variants of the CCL2 protein have been purified and compared for their distinctive biophysical characteristics employing a combination of biophysical techniques. Furthermore, the varying binding affinities of CCL2 with different glycosaminoglycans (GAGs) have been explored. Additionally, the ability of CCL2-WT/M proteins to directly interact with widely known immuno-modulatory flavonoids such as catechins and oxyresveratrol have been evaluated, which interfere in the CCL2-CCR2 axis mediated cellular signalling and inhibit the CCL2-guided migration of macrophages. Moreover, the molecular interactions between CCL2 and suramin, a GAG mimetic has been elucidated. These studies are detailed in the respective chapters (Chapter 1-5) of the thesis and are described as follows. Chapter 1 provides a comprehensive exploration of the immune system and its constituent elements, namely the innate immune system and the adaptive immune system. It dwells extensively upon the existing scientific literature regarding chemokines, encompassing their categorization, structural intricacies, chemokine receptors, and the significance of chemokine-receptor interactions in governing biological functions. Furthermore, the chapter offers a thorough examination of CC chemokines, exploring the structural and functional characteristics of different CC family members including a detailed scrutiny of the physiological and structural attributes of CCL2 chemokine. Additionally, this chapter offers detailed insights into glycosaminoglycans (GAGs), their classification, and their interaction with CCL2 in regulating monocyte recruitment. The chapter also details the involvement of CCL2 in various pathological conditions and outlines its clinical relevance. The final section of Chapter 1 provides an extensive overview of small molecules (both natural and synthetic in origin), potent enough to act as a CCL2 inhibitor, their categorization, chemical structures, biological functions, and clinical significance. Chapter 2 is specifically dedicated to deciphering the structural characteristics of the engineered monomer (P8A) of CCL2 (CCL2-M) chemokine, and its binding interactions with glycosaminoglycans (GAGs). Given the absence of structural data pertaining to monomeric variants of CCL2 proteins, this chapter aims to uncover the structural and in vitro functional attributes of CCL2-M. The structural and dynamics features have been dissected using an array of 2D and 3D based NMR experiments in conjunction with molecular dynamics (MD) simulations. The detailed biophysical analysis disclosed that the engineered variant exists exclusively in a monomeric conformation and displays comparable dynamic alterations within the microsecond to millisecond timescales. Additionally, this chapter offers molecular level comparison of the distinct interactions of various GAGs with CCL2 chemokine using NMR spectroscopy and isothermal calorimetry. In summary Chapter 2 depicted the molecular contacts and binding affinities of these complex interactions apart from structural, dynamics and functional features of CCL2-M. Chapter 3 delineates the binding characteristics between catechins and CCL2 protein. The molecular interactions between catechins and CCL2 were investigated using a comprehensive array of methodologies, encompassing steady-state fluorescence spectroscopy, transwell migration assays, NMR spectroscopy, molecular docking, and molecular dynamic simulations. Fluorescence quenching experiments and transwell migration assay revealed that the catechin possessing gallate moiety (Epicatechin gallate, ECG; Epigallocatechin gallate, EGCG) could only attenuate CCL2-induced macrophage migration and the binding affinity of EGCG (Kd = 22 ± 4 μM) is ∼4 times higher than that of the ECG complex (Kd = 85 ± 6 μM). NMR based titration experiment demonstrated that ECG and EGCG bind to CCL2 on a pocket comprising the N-terminal, β0-sheets, and β3-sheets. The NMR-based perturbations identified a significant overlap of the CCL2 binding surface for EGCG/ECG and CCR2 receptor which is in line with the observed inhibition of macrophage migration in response to CCL2- EGCG/ECG interactions. Furthermore, the effect of structural stereospecificity was also demonstrated based upon the computational studies performed using methylated counterparts of EGCG. MD simulation analysis evidenced that the molecular specificity/stability of CCL2– catechin complexes is regulated by multiple factors, including the stereospecificity, number of hydroxyl groups on the annular ring-B, orientation of the carbonyl group, and methylation of the galloyl ring. Conclusively, Chapter 3 provides the comprehensive structural and functional insights about CCL2–catechin interaction providing compelling evidence to deliberate EGCG as nutraceuticals and as parent molecules for formulating therapeutic immunomodulatory agents to regulate chemokine-arbitrated inflammatory disorders. Chapter 4 of the thesis delineates a comparative study on the binding interactions of stilbenes and CCL2 protein. Building upon Chapter 3, which revealed specific high-affinity binding of catechins to CCL2, the current chapter screened potential glycosidic flavonoid candidates from the stilbene subgroup against CCL2 monomer using Autodock Vina. Compounds exhibiting the highest binding energies were screened and selected according to their ADMET properties which ultimately identified oxyresveratrol (ORS) as a promising CCL2 binding partner. To elucidate the molecular interactions between ORS and CCL2 protein, a suite of techniques including steady state fluorescence spectroscopy, transwell migration assay, NMR spectroscopy, molecular dynamic simulation, and binding free energy calculation were employed. Fluorescence quenching experiments revealed ORS interacts with both monomeric and dimeric forms of CCL2, demonstrating similar binding affinities in the micromolar range (CCL2-M, Kd = 7.7 ± 1 μM; CCL2-WT, Kd = 5.98 ± 1 μM). Additionally, the transwell migration assay demonstrated that ORS inhibits CCL2-M/WT guided migration of macrophages. NMR experiments and molecular docking studies unveiled the molecular interactions and binding sites of ORS on the CCL2 protein. Owing to the QSAR advantages of natural ligand derivatives, 27 structural derivatives of ORS were screened leading to identification of two derivatives (ORS10 and ORS16) from which ORS16 was ultimately identified as a potential ORS derivative through MD studies as a potential anti-inflammatory candidate. Chapter 4 provides novel insights into the binding of ORS and its structural derivatives (ORS10 and ORS16) to CCL2, offering valuable information regarding the molecular mechanisms of ORS-chemokine interactions in various immunomodulatory processes. Chapter 5 of the thesis demarcated the binding studies of a potential GAG mimetic suramin with CCL2. The binding surface of chemokine has two distinctly dedicated pockets for GAG and receptor binding. Suramin is an anti-parasitic, anti-inflammatory compound that shares a structural similarity with heparin, a ubiquitous glycosaminoglycan which sumptuously binds to chemokines and induces leukocyte migration and oligomerization. Taking this into consideration, the current chapter explores the affinity of suramin to act as a GAG mimetic by studying the interaction pattern of suramin with CCL2 chemokine using an array of biophysical and cell-based studies. Fluorescence quenching experiments demonstrated that SUR interacts with the monomeric variant of CCL2 with nanomolar range (Kd = 110 ± 9 nM). Fluorescence lifetime study revealed that the CCL2-SUR complexation disturbs the overall electronic environment of the tryptophan (W59) of the CCL2. Moreover, a noticeable reduction in the secondary structure pattern of CCL2, indicated by CD spectroscopy affirms these findings. In addition, findings from the NMR experiments and molecular docking studies unveiled the molecular interactions and binding sites of SUR distributed on the N-terminal, 1st loop, and β2/β3-sheet, 3rd loop of the CCL2 protein. Furthermore, transwell migration assays demonstrated that SUR effectively inhibits the CCL2-M-mediated migration of macrophages. In summary, Chapter 5 offers a novel perspective on the interaction between the GAG mimetic Suramin and CCL2 chemokine, shedding light on its immunomodulatory effects. In summary, the present research work deciphered the structural facets of engineered CCL2 monomer, CCL2-P8A chemokine. The monomeric variant of CCL2 has shown akin differences in dynamic features and receptor/GAG-binding surfaces although the overall structure of wild-type and engineered proteins are well conserved. Moving further, molecular/atomic-level interaction of CCL2 with different heparin derivatives, nutraceuticals (flavonoids), and an FDA approved drug suramin revealed their significant interaction affinities with CCL2 and highlighted the potential usage of flavonoids/flavonoid derivatives and suramin as protein-receptor/GAG inhibitors respectively. These molecular interactions with CCL2 and small molecules warrant further studies on other subtypes of chemokines along with in vivo studies to decipher the key mechanisms which attenuate the cell signaling processes arbitrated through CCL2-CCR-GAG axis.