SSB-2548 Attenuates C-X-C chemokine receptor type 4 (CXCR-4) Activation to Induce Apoptosis; An in silico Guided invitro Validation in Acute Myeloid Leukemia cells

doi:10.21203/rs.3.rs-3767578/v1

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SSB-2548 Attenuates C-X-C chemokine receptor type 4 (CXCR-4) Activation to Induce Apoptosis; An in silico Guided invitro Validation in Acute Myeloid Leukemia cells

https://doi.org/10.21203/rs.3.rs-3767578/v1

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Background and Aim:

The role of C-X-C chemokine receptor type 4 (CXCR-4) in chemotherapy resistance remains crucial in promoting proliferation, invasion and progression in Acute Myeloid Leukemia (AML) cells. This study aims to screen and investigate a potential lead candidate as a therapeutic agent targeting CXCR-4 in AML cells.

Methods

Diversity-based virtual screening process using Autodock-vina was employed to screen approximately 8,50,000 compounds from the ChemBridge-small molecule database. The binding stability and dynamics was investigated through GROMACS-based molecular dynamics simulations and RMSD. AML cells (THP-1, HL-60 and SKM-1cell cell lines) was used to assess proliferation CXCR-4 expression and apoptosis induction were measured using flow cytometry and trans-endothelial migration was assessed using calorimetric method in AML cells. The ADME properties were predicted using SwissADME server.

Results

The computational evaluations revealed SSB-2548 as a lead candidate that binds stably to CXCR-4. Molecular dynamics simulations provided detailed insights into the conformational changes of the SSB-2548/CXCR-4 complex. The compound inhibited the THP-1, HL-60 and SKM-1cell proliferations with GI₅₀ values of 84.57 nM, 41.30 nM and 120.50 nM respectively. SSB-2548 decreased the trans-endothelial migration and CXCR-4 expression in while inducing early and late phase apoptosis in all three AML cell types. ADME predictions indicated a favorable lead-likeness, gastrointestinal absorption and lack of notable toxicity.

Conclusion

Computational assessments identified SSB-2548 as a novel CXCR-4 inhibitor. Invitro evaluations proved this lead compound effective against AML cells. These findings lay the groundwork for future investigations positioning SSB-2548 as a candidate for the development of targeted therapies against AML.

AML

ADME predictions

Apoptosis

Computational high throughput screening

CXCR-4

Molecular dynamic simulation

Computational High throughput analysis identified SSB-2548 as a novel lead compound against CXCR-4.
The MD simulations demonstrate stable binding between SSB-2548 and CXCR-4 over the simulated time, indicating a robust interaction with the hydrophobic pocket in the Extracellular Loop (ECL) region of CXCR-4.
Demonstrated invitro efficacy of SSB-2548 suggested it as a novel lead small molecule candidate represents a promising new class of therapeutic agents for AML treatment with potentiality to be developed against AML.
Favorable ADME predictions like absence of notable toxicity, including the lack of PAINS properties indicate that SSB-2548 possesses good lead-likeness, suggesting its potential suitability for further development as a drug candidate.

Leukemia is a broader term for cancer of blood cells, affecting young and old populations globally [1]. Acute myeloid leukemia (AML), the hematopoietic, aggressive, proliferative malignancy, is heterogeneous in nature. It affects the myeloid cells of the bone marrow (BM) and is most commonly seen in adults and rarely in children [2]. It is a clonal disorder characterized by the accumulation of excessively proliferated hematopoietic stem cells (HSCs) and their progenitors which pile up as abnormally differentiated immature myeloid cells in the BM [3]. Both the endosteal landscape and the blood vessels in the BM environment, provide signals to assist functioning of normal HSCs as well as leukemia initiating stem cells (LSCs) [4]. The LSCs are found abundant in AML and are capable of endless self-renewal leading to buildup of leukemic stem cell blast progenitors. The rapid expansion of immature myeloid progenitor in the BM, blood, and hematopoietic regions lead to massive changes in the endosteal niche, loss and leaking of vasculature causing hypoxia, creating pro-carcinogenic microenvironment to allow growth of AML cells [5]. The normal hematopoietic cells are sacrificed to aid the expansion of carcinogenic cells and this eventually hampers chemotherapy-induced AML cell death [6].

The relapse and recurrence of AML cells that remain unhindered by chemotherapy could be attributed to the involvement of chemokine receptor 4 (CXCR-4) which is found elevated in the LSCs in the BM microenvironment [7].CXCR-4 which was first discovered as a co-receptor facilitating the entry of human immunodeficiency virus (HIV) into the human body, is a part of the chemokine receptor family of transmembrane G-protein coupled receptors [8]. Chemokines are small peptides secreted by many cells like immune cells, stromal cells, tumor cells etc. CXCR-4 is widely expressed in all types of cells including hematopoietic cells. Leukemic blasts show a high concentration of CXCR-4 attached to its ligand CXCL12 (also called stromal cell-derived factor 1 - SDF1). Several studies have reiterated the involvement of the chemokine axis CXCR-4 - SDF-1/CXCL12 in activating a cascade of intracellular pathways, like Janus kinase (JAK), JAK-STAT (signal transducers and activators of transcription), phosphatidylinositol-3-kinase (PI3K), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), RAS-MAPK, PI3K-AKT, and mammalian target of rapamycin (mTOR) [2, 3]. This causes chemotaxis, gene expression, cell survival, cell trafficking, cell proliferation resulting in tumor growth, progression and metastasis.

Many studies have established the role of CXCR-4 - SDF-1/CXCL12 in the “cross-talk” between leukemia cells and BM. The metabolic cross-talk of AML LSCs and BM stromal cells in the endosteal niche due to the high metabolic plasticity of AML cells help them to survive (reactive oxygen species) ROS-induced apoptosis [9–11]. This results in leukemia proliferation, extramedullary migration, infiltration, adhesion, and chemoresistance. The involvement of the CXCR-4-CXCL12 axis in homing to and retention in BM of leukemic cells has been proven in many researches and their interaction causes retention of AML blasts affecting the prognosis [2, 12, 13].

AML treatment options involve chemotherapy, radiation therapy, combination of chemotherapy with stem cell transplant etc. Use of CXCR-4 antagonists as monotherapy or as a prelude setup before hematopoietic cell transplant therapy is highly recommended owing to the aggressive nature of the disease [12, 13]. One predominant reason to target CXCR-4-CXCl12 axis in AML is that its disruption will not only inhibit pro-survival signaling but also mobilize LSCs from the protected BM microenvironment to the vasculature where they could be subjected to chemotherapeutic agents and this strategy is called “chemo-sensitization.” In addition to this, the CXCR-4 inhibitors have cytotoxic effects, and can induce apoptosis in the cells that express the receptor. The antagonists can inhibit migration of AML cells induced by SDF-1 and impede several intracellular pathways, thereby reversing the protective effect of stromal cells on AML cells [2, 14].

CXCR-4 antagonists include several small molecule CXCR-4 inhibitors, short peptide-like CXCR-4 antagonists, antibodies to CXCR-4, antibody drug conjugates, and CXCL12 antagonists. On broader terms, the first two antagonists have cationic regions that can bind efficiently to the anionic extracellular region of CXCR-4. A few monoclonal antibodies have been isolated targeting the chemokine CXCR-4-CXCL12 axis that employ antibody-dependent phagocytosis, or antibody-dependent cell-mediated toxicity, facilitating the elimination of AML cells. The CXCL12 antagonists disrupt the chemokine axis and make the malignant cells prone to chemo-sensitization. Antibody drug conjugates, radionuclide therapy, use of nanomaterials that target the CXCR-4 has been put up in trials and shown good results [14, 15].

However, targeting CXCR-4 needs a detailed understanding of the structure of CXCR-4. It comprises 352 amino acid residues with one amino terminal (N-terminal domain), seven transmembrane domains (TM domain), three extracellular loops (ECL), three intracellular loops (ICL), and one carboxyl terminal (C-terminal domain). CXCR-4 is a homodimer and its binding cavity is larger and closer to ECL, while its ligand CXCL12 is bound to N-terminal and ECL. Use of small molecules to target the cationic-anionic interactions respectively between the CXCR-4 antagonists and extracellular domain of CXCR-4 are studied widely and as many as 20 chemical compounds have been identified, but currently only one compound, plerixafor is approved by FDA (Food and Drug Administration), and the rest are in clinical trials [16]. Therefore, in order to identify more targeted compounds with improved specificity and efficacy against CXCR-4, this study aims in screening the ChemBridge small molecule database to identify potential lead compound using a multifaceted approach.

2.1 Materials

All reagents and chemicals were obtained from Sigma Aldrich (St. Louis, MO, USA). HUVEC, THP-1, TF-1, HL-60 and SKM-1 cell lines were from ATCC (American Type Culture Collection, Rockville, MD, USA). Annexin V assay kit and Trans-endothelial migration assay kit were from Merck Millipore (Burlington, MA, USA). FITC tagged extracellular anti-CXCR-4 polyclonal antibody (#ACR-014-F) was from ThermoFisher Scientific (Waltham, MA, USA).

2.2 Methods

2.2.1 Structure retrieval and high-throughput virtual screening

The crystal structure of CXCR-4 was acquired from the PDB database (3OE0) and underwent preprocessing using Discovery Studio Visualizer. This involved eliminating crystal waters and incorporating polar hydrogens. The SwissPDB viewer was employed to address missing side chains and atoms. A docking grid box, based on the protein-protein interaction interface, was generated with dimensions of 20 units on all sides. High-throughput Virtual Screening of the ChemBridge library, limited to molecules with a molecular weight (MW) between 350 to 750 Da, was executed using the D-HTVS module from SiBioLEAD [17]. The examination of protein-ligand interactions was carried out using the PLIP analysis plugin, and the outcomes were visualized through Discovery Studio Visualizer. Known CXCR-4 inhibitors were obtained from the PubChem database in SDF format and converted into SYBYL-TRIPOS (mol2) format using Discovery Studio Visualizer.

2.2.2 Molecular Dynamic Simulation

The Molecular Dynamics (MD) simulation of CXCR-4 in conjunction with the target ligand was computed via an MD simulation server utilizing the GROMACS simulation package, accessible at www.sibioled.com. The ligand topology was generated using AMBERTOOLS and the ACPYPE package, and the simulation system was parameterized with the OPLS/AA force field [18]. Initially, the protein-ligand complex found itself immersed in a triclinic box containing Simple Point Charge (SPC) water for hydration. To replicate physiological conditions, the simulation system was neutralized using NaCl as counterions, supplemented with an additional 0.15M NaCl salt. The simulation system, encompassing the protein-ligand complex, SPC water, and salt ions, underwent energy minimization for 5000 steps using the steepest descent method. The simulation system was equilibrated for 300 ps under constant temperature and pressure, utilizing the NVT/NPT protocol. The MD run extended for 200 ns using a leap-frog integrator, capturing trajectory frames at every 20 ps. GROMACS in-built trajectory analysis scripts were employed for the analysis of simulation trajectories, and the results were visualized using xmgrace. The Gibbs binding free energy (ΔG binding) of the protein-ligand complexes was computed employing the GMX_MMPBSA method [19]. Gibbs binding free energies were generated using trajectory frames saved at every 4 ns over the course of 200 ns.

2.2.3 Cell culture and anti-proliferation assay

AML cells were cultured in RPMI-1640 medium with 10% FBS, 100 U/ml of penicillin, and 100 U/ml streptomycin as per standard protocol. Cell proliferation inhibition was assessed using MTT assay as described elsewhere [20]. Percentage proliferation inhibition was analyzed and GI₅₀ values were presented using GraphPad Prism 6.0 software.

2.2.4 CXCR-4 inhibition assay

THP-1, HL-60 and SKM-1 cells at a concentration of 1X 10⁵ / ml/ well, were seeded in 24 well plate. Following SSB-2548 treatments, cells were incubated for 24 h. Control cells were treated with DMSO alone. Following the incubation, cells were washed twice with ice-cold sterile PBS and resuspended in HBSS permeabilization buffer. Control and treated cells were added with 2.5 µg/ml FITC coated anti -CXCR-4 antibody and further incubated for 30 min in dark. After a couple of washes with ice-cold PBS, the cells were resuspended in the HBSS buffer and 10,000 events acquired in a Guava easyCyte™ flow cytometer. The data were analyzed with InCyte Software from Millipore, (Burlington, CA USA) and respective percentage positive cells were presented with respect to their controls.

2.2.5 Tumor cell trans-endothelial migration assay

The assay was carried out as described elsewhere [21] using kit method as per the manufacturer's instructions. Briefly, HUVEC cells at a concentration of 1x10⁵ were grown on the provided migration inserts until confluent. AML cells were allowed to starve overnight in serum-free media and replaced in the inserts. Different concentrations of SSB-2548 was added to the inserts and then transferred to wells that contain 50 ng/ml Hepatocyte growth factor (HGF) as chemo attractant and allowed to migrate across the membrane for 12 hours in a CO₂ incubator. The migration inserts were gently swabbed, stained and eluted with kit components. The eluent was transferred to a 96-wells stain quantification plate and absorbance read at 540–570 nm. Percentage migration inhibition was analyzed and presented using GraphPad Prism 6.0 software.

2.2.6 Apoptosis detection assay

All three AML cells were treated with different doses of SSB-2548 along with DMSO controls and incubation for 48 h. Apoptosis was analyzed by using Annexin V detection kit as described elsewhere [22]. Percentage of early and late phase apoptotic populations in comparison to respective controls were presented.

2.2.7 Absorption, Distribution, Metabolism, and Excretion (ADME) predictions

SwissADME prediction tool is a web-based platform designed for the prediction of various molecular properties and pharmacokinetic parameters, including lipophilicity, water solubility, drug-likeliness, and bioavailability score. SwissADME offers predictions for Absorption, Distribution, Metabolism, and Excretion (ADME) properties of small molecules. For predicting the properties, target compound(s) were uploaded on to the SwissADME server (http://www.swissadme.ch/) [23] and properties were predicted.

2.2.8 Statistical analysis

GraphPad Prism 6.0 (La Jolla, USA) was used to analyze data for statistical evaluations. Results were expressed as the mean ± SD. GI₅₀ values were calculated using a non-linear regression fit model with variable slope. p values of ≤ 0.05 (*) were considered significant.

3.1 High throughput virtual screening of small molecules against CXCR-4.

Toward identifying new small molecules that specifically target the extracellular region (ECL) of CXCR-4 and inhibit the downstream targets, experimental structure of CXCR-4 bound to a peptide antagonist was retrieved (PDBID:3OE0) and analyzed (Fig. 1a & b). Analysis of the 3D structure shows the known peptide antagonist CVX15 binds at the ECL region of CXCR-4 (Fig. 1b), and the protein-ligand analysis reveals the above-said peptide interacts with crucial amino acid residues, including Arg188, Gln200, and Thr117 (Fig. 1c). A high-throughput virtual screen (HTVS) of the ChemBridge library molecules with molecular weight between 350–750 Da, against the ECL region of CXCR-4 was performed. The top compounds were ranked based on their docking scores, which are a measure of their predicted affinity for binding to CXCR-4 (Fig. 2a). The compound SSB-2548 showed a binding energy of -10.4 kcal/mol (Fig. 2b), and protein-ligand interaction profiling shows, it binds exactly at the ECL pocket of the CXCR-4 (Fig. 2c), and hence among the top compounds, compound SSB-2548 was selected for further analysis (Fig. 2d). In addition, the docking results show it interacts with amino acids, including Asp187, Leu41, Asn33, Arg30, Lys282, Ser285, and Asn37 at the ECL region of CXCR-4 (Fig. 2d).

3.2 Atomistic molecular dynamic simulation of SSB-2548 bound CXCR-4

In order to assess the binding stability and the dynamics of SSB-2548 in complex with CXCR-4, a fully-solvated atomistic molecular dynamic (MD) simulation of CXCR-4::SSB-2548 complex was performed for 200ns. MD simulation was conducted using the GROMACS simulation software through a web-based application (www.sibiolead.com). During this time, the system was allowed to evolve and simulate the dynamics of SSB-2548 bound to CXCR-4. The simulation snapshots taken at different timepoints of simulation were compared. This visual analysis indicates that SSB-2548 remains stably bound to CXCR-4 at the predicted ECL region throughout the simulation (Fig. 3a). The Root Mean Square Deviation (RMSD) of the ligand (SSB-2548) was calculated throughout the simulation. The RMSD value was less than 0.2 nm, except for a few occasional fluctuations, suggesting that the binding of SSB-2548 to CXCR-4 is stable. The results show a stable H-bond pattern for SSB-2548 throughout the simulation, indicating that the ligand maintains consistent interactions with the receptor.

3.3 Gibbs binding energy estimation and comparison of SSB-2548 with other known CXCR-4 inhibitors

To identify the binding stability of SSB-2548, a detailed analysis was conducted utilizing 200 ns simulation trajectories. The Gibbs binding free energy, calculated through the Molecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) method, was determined based on 50 frames extracted from the simulation duration. The gmx_MMPBSA tool facilitated the estimation, indicating a highly favorable and robust binding with a calculated binding energy of -28.63 kcal/mol (Fig. 4a).

To gain further insights into the binding efficacy of SSB-2548 with CXCR-4, its predicted docking energy was juxtaposed with that of known CXCR-4 inhibitors. This comparative assessment indicated that SSB-2548 exhibits superior binding affinity for CXCR-4 than others (Fig. 4b).

3.4 SSB-2548 effectively controlled AML cell proliferations.

Before assessing the effects of the compound on cancer cells, the maximum tolerated dose (non-toxic concentrations) of the compound in the non-cancerous Vero cells was determined. No change in cell viability of Vero cells was found up to 1000 nM of SSB-2548 (Fig. 5a). A reduction in percentage cell viability was observed from 1500 nM concentration (Fig. 5a). A dose-dependent decrease in the cell viability of THP-1, HL-60, and SKM-1 cells was noted with SSB-2548 treatments. The compound inhibited THP-1 cell proliferation with a GI₅₀ value of 84.57 nM (Fig. 5b). The compound had GI₅₀ values of 41.30 nM and 120.50 nM in HL-60 and SKM-1 cell proliferations, respectively (Fig. 5b). The near GI₅₀ concentrations, i.e., 85 nM for THP-1 cells, 40 nM for HL-60 cells, and 120 nM for SKM-1 cells, were used for further evaluations.

3.5 SSB-2548 reduced CXCR-4 expression in AML cells

DMSO-treated THP-1, HL-60, and SKM-1 cells had 43.11%, 39.75%, and 48.47% positive population of CXCR-4, respectively (Fig. 6). Treatment of SSB-2548 reduced the CXCR-4 positive population to 12.63%, 08.69%, and 14.58% in THP-1, HL-60, and SKM-1 cells, respectively (Fig. 6).

3.6 SSB-2548 inhibited trans endothelial migration of AML cells and promoted early and late phase apoptosis

SSB-2548 suppressed the trans-endothelial migration of THP-1, HL-60, and SKM-1 cells across the HUVEC membrane by 35.0%, 54.66%, and 43.35%, respectively (Fig. 7a). Treating THP-1 cells with 85 nM SSB-2548 induced 32.65% early and 10.94% late apoptotic cell populations (Fig. 7b). HL-60 cells, subjected to 40 nM SSB-2548, exhibited 30.64% and 18.86% early and late apoptotic cell populations, respectively (Fig. 7b). Similarly, in SKM-1 cells, 24.56% and 11.43% of respective early and late-phase apoptotic populations were observed upon treatment with 120 nM SSB-2548.

3.7 ADME prediction for SSB-2548 showed favorable drug-likeness.

Toward identifying the drug-likeness of the identified lead compound, SSB-2548, the physicochemical properties of the lead compound were predicted using the SwissADME server. Results show that the Gastrointestinal (GI) absorption, and Lipinski rules are in the acceptable range. There is no indication of PAINS properties, and lead likeness was good for SSB-2548 (Fig. 8).

The significance of the CXCR-4:CXCL12 axis in the homing to and retention in the bone marrow of leukemic cells has been extensively demonstrated in various studies [3]. Their interaction plays a crucial role in the retention of AML blasts, thereby influencing the prognosis. The CXCR-4:CXCL12 axis is known to stimulate diverse downstream signaling pathways that regulate chemotaxis, gene transcription, cell survival, and proliferation. The binding of CXCL12 to CXCR4 plays a significant role in cell proliferation. Upon binding, CXCL12 induces a three-dimensional conformational change in CXCR4, initiating a signaling cascade. This molecular interaction triggers the exchange from GTP to GDP, leading to the dissociation of the Gα subunit from the Gβ/Gγ dimer. This dissociation activates downstream signaling pathways that contribute to cellular processes, including cell proliferation. The CXCL12/CXCR4 axis is known to be involved in various cellular functions, and its dysregulation can have implications in conditions such as cancer and immune responses [24]. The association of CXCL12 with cancer cells is predicted to have two immunological consequences. Previous studies have demonstrated that targeting CXCR-4 with a cyclic peptide (CVX15) effectively blocked the activation of CXCR-4. Experimental models have shown that CVX15 targets the hydrophobic site in CXCR4, i.e., the extracellular pocket where CXCL12 binds. This binding leads to conformational changes in CXCR4, triggering downstream signaling pathways that are associated with CXCR4 activation, and in this way providing a potential avenue for therapeutic intervention [25].

In this investigation, a comprehensive approach was employed to identify a novel class of lead molecules against CXCR-4. The exploration involved a diversity-based virtual screening process utilizing Autodock-vina, where approximately 850,000 compounds were virtually screened from the ChemBridge database. The screening specifically targeted the hydrophobic pocket located in the extracellular region of the CXCR-4 protein. Studies on the activation mechanism of CXCR-4 underscored the significance of the extracellular region and its associated hydrophobic pocket in the activation and functioning of CXCR-4 [25, 26].

CXCL12 is known to bind to CXCR-4 via the ECL region of CXCR-4. Inhibitors targeting this region attenuated CXCR-4 activation [27]. According to the docking scores, based on the high-throughput virtual screening, compound SSB-2548 showed a favorable binding energy. Since docking energy alone cannot predict the potential lead compound, atomistic molecular dynamic simulation was conducted. Studies indicate that MD simulations when conducted in a condition close to physiological situations, including water and salt concentrations, the false positive rate could be considerably low [28]. MD simulation of SSB-2548 bound to CXCR-4 showed a stable binding throughout the 200ns simulation. This indicates that the predicted ligand has comfortable binding to CXCR-4 without any potential clashes within the binding site. Furthermore, Gibbs binding free energy estimation from the simulation trajectories indicated a strong binding affinity, suggestive of stable binding. We suggest that SSB-2548 holds the potential to inhibit CXCR-4 significantly at lower concentrations too, positioning it as a lead candidate for further in-depth analysis and consideration in the development of targeted therapies. Overall, the MD simulation results suggested avid and stable binding of SSB-2548 to CXCR-4. Studies have shown that compounds targeting the hydrophobic site at the extracellular region in CXCR4 inhibited the downstream signaling pathways associated with CXCR4 activation [29]. Based on these prior studies, it is noteworthy that the identified compound SSB-2548 binds at the hydrophobic site on the extracellular domain of the CXCR-4. This suggests the potential of SSB-2548 to inhibit downstream activities of CXCR-4 [25]. The remarkable binding stability and energy estimate highlight the therapeutic potential of SSB-2548 in reducing CXCR-4 activation. Over and above, comparison of SSB-2548 with other known CXCR-4 inhibitors clearly showed that SSB-2548 has superior binding over other known inhibitors. The significance of SSB-2548 as a novel therapeutic agent against CXCR-4 in controlling cell proliferation in AML cells was emphasized by its promising characteristics. Moreover, the effective drug concentration for inhibiting the AML cell proliferations were well below the non-toxic dose of the compound.

SSB-2548 demonstrated a notable impact by inhibiting the trans-endothelial migration of AML cells. Furthermore, its influence extended to the induction of early and late-phase apoptosis in these cells. The inhibition of trans-endothelial migration suggests a potential interference with the ability of AML cells to traverse endothelial barriers, which is crucial for their dissemination and infiltration. Moreover, the observed promotion of both early and late-phase apoptosis indicates a multifaceted effect on cell survival mechanisms[30]. Early apoptosis is often associated with the initiation of programmed cell death, while late-phase apoptosis signifies advanced stages of cellular demise [31]. This dual impact on migration and apoptosis underscores the potential therapeutic relevance of SSB-2548 in the context of AML, suggesting its ability to impede cell mobility and induce programmed cell death, which are critical aspects in addressing the pathology of AML. CXCR4 plays a pivotal role in the process of trans-endothelial migration, particularly in the context of immune responses and various pathological conditions. When CXCL12 binds to CXCR4, it triggers chemotaxis, attracting cells expressing CXCR4 towards the source of the chemokine. This chemotactic response is crucial for the directed movement of cells through tissues and across endothelial barriers [32]. Since the lead molecule SSB-2548 identified here inhibited trans-endothelial migration, it suggests the compound to act via CXCR4.

ADME predictions have indicated that SSB-2548 exhibits good lead-likeness with no notable toxicity, including the absence of PAINS (Pan Assay Interference Compounds) properties. Additionally, predictions regarding gastrointestinal (GI) absorption have suggested a high likelihood, providing a robust foundation for advancing SSB-2548 as a CXCR-4 inhibitor. These favorable ADME predictions contribute to the potential of SSB-2548 as a viable candidate for further development, emphasizing its suitability for progressing into preclinical and clinical studies.

Despite the advancements in computational evaluations and invitro studies that provide valuable insights, further research in in vivo models and clinical trials are necessary to determine the safety, efficacy, and therapeutic potential of CXCR-4 to be further developed as effective chemotherapeutic in AML treatment.

Aberrant expression and activation of CXCR-4 have been associated with the pathogenesis of AML. Leukemic cells often exploit the CXCR-4 pathway to stay in the protected bone marrow niche, and they evade the effects of chemotherapy to promote disease progression. The identification of lead compound SSB-2548, that binds to the hydrophobic pocket in the Extracellular Loop (ECL) region of CXCR-4 represents a promising avenue for developing novel anticancer agents that disrupt the interaction between leukemic cells and the bone marrow microenvironment, potentially leading to more effective treatments for AML. However, further preclinical and clinical validations are warranted for further developments.

Statements and Declarations :

Funding: The author extends his appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through small group Research Project under grant number RGP1/310/44

Ethics approval statement: This is an experimental study. The KKU Research Ethics Committee has confirmed that no ethical approval is required.

Consent to Participate and Publish: the author accepted responsibility for the entire content of this submitted manuscript and approved submission.

Authors Contributions: Conceptualization, methodology, validation, formal analysis, writing, and funding acquisition A.A.D. the author has agreed to the published this version of the manuscript.

Acknowledgements: Authors express their gratitude to SMARTBIO LABS, Chennai-78, Tamil Nadu, India and Si-BIOLEAD, Little Rock, Arkansas, USA for the help rendered in this study.

Conflict of Interest: The authors declare that they have no conflict of interest.

Competing Interests: The authors have no relevant financial or non-financial interests to disclose

Data Availability: All data are used in this manuscript is available with the author and can be provided on reasonable request for noncommercial purposes.

Patient consent statement: This study does not involve human patients and therefore this section does not apply.

Permission to reproduce material from other sources: Not applicable.

Clinical trial registration: Not applicable

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SSB-2548 Attenuates C-X-C chemokine receptor type 4 (CXCR-4) Activation to Induce Apoptosis; An in silico Guided invitro Validation in Acute Myeloid Leukemia cells

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Abstract

Background and Aim:

Methods

Results

Conclusion

Figures

Research Highlights

1. Introduction

2. Materials and Methods

2.1 Materials

2.2 Methods

2.2.1 Structure retrieval and high-throughput virtual screening

2.2.2 Molecular Dynamic Simulation

2.2.3 Cell culture and anti-proliferation assay

2.2.4 CXCR-4 inhibition assay

2.2.5 Tumor cell trans-endothelial migration assay

2.2.6 Apoptosis detection assay

2.2.7 Absorption, Distribution, Metabolism, and Excretion (ADME) predictions

2.2.8 Statistical analysis

3. Results

3.1 High throughput virtual screening of small molecules against CXCR-4.

3.2 Atomistic molecular dynamic simulation of SSB-2548 bound CXCR-4

3.3 Gibbs binding energy estimation and comparison of SSB-2548 with other known CXCR-4 inhibitors

3.4 SSB-2548 effectively controlled AML cell proliferations.

3.5 SSB-2548 reduced CXCR-4 expression in AML cells

3.6 SSB-2548 inhibited trans endothelial migration of AML cells and promoted early and late phase apoptosis

3.7 ADME prediction for SSB-2548 showed favorable drug-likeness.

4. Discussion

Conclusion

Declarations

References

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