GPC-100, a Novel CXCR4 Antagonist, Improves in Vivo Hematopoietic Cell Mobilization When Combined With Propranolol
Devki D. Sukhtankar, Juan José Fung, Mi-na Kim,Thomas Cayton, Valerie Chiou, Niña G. Caculitan, Piotr Zalicki, Sujeong Kim, Yoonjung Jo,SoHui Kim, Jae Min Lee,Junhee Choi, SeongGyeong Mun, Ashley Chin, Yongdae Jang, Ji Yeong Lee, Gowoon Kim, Eun Hee Kim, Won-Ki Huh, Jae-Yeon Jeong, Dong-Seung Seen, Pina M. Cardarelli
Abstract
Autologous Stem Cell Transplant (ASCT) is increasingly used to treat hematological malignancies. A key requisite for ASCT is mobilization of hematopoietic stem cells into peripheral blood, where they are collected by apheresis and stored for later transplantation. However, success is often hindered by poor mobilization due to factors including prior treatments. The combination of G-CSF and GPC-100, a small molecule antagonist of CXCR4, showed potential in a multiple myeloma clinical trial for sufficient and rapid collection of CD34+ stem cells, compared to the historical results from the standards of care, G-CSF alone or G-CSF with plerixafor, also a CXCR4 antagonist. In the present study, we show that GPC-100 has high affinity towards the chemokine receptor CXCR4, and it potently inhibits β-arrestin recruitment, calcium flux and cell migration mediated by its ligand CXCL12. Proximity Ligation Assay revealed that in native cell systems with endogenous receptor expression, CXCR4 co-localizes with the beta-2 adrenergic receptor (β2AR). Co-treatment with CXCL12 and the β2AR agonist epinephrine synergistically increases β-arrestin recruitment to CXCR4 and calcium flux.
Introduction
Multiple myeloma (MM) is a leading hematological malignancy with an estimated 34,920 cases in the United States and approximately 588,161 cases worldwide each year [1]. Autologous Stem Cell Transplant (ASCT) is integral to the overall management of MM in eligible patients and has improved the anti-cancer response and survival compared to conventional chemotherapy [2–5]. The success of ASCT relies on harvesting a sufficient number of hematopoietic stem cells (HSC), which are predominantly obtained by mobilizing the HSCs from the bone marrow (BM) into the peripheral blood (PB) [6, 7]. In humans, HSCs are phenotypically characterized by the expression of CD34. A minimum of 2 x 106 CD34+ cells/kg are essential for the HSC harvest, whereas the optimal number for improved engraftment and survival is >5–6 x 106 CD34+ cells/kg [8, 9]. Granulocyte-colony stimulating factor (G-CSF) is a clinical standard of care for HSC mobilization [10]. However, G-CSF fails to mobilize optimal number of HSC in at least 40–50% MM patients [10, 11]. Some patients are treated with the combination of G-CSF and a small molecule CXCR4 antagonist plerixafor (AMD3100) [10]. Despite this combination treatment, 15–35% MM patients do not mobilize a sufficient number of cells [10, 12]. In a recent phase 3 clinical study, the combination of G-CSF and motixafortide, a peptide inhibitor of CXCR4, mobilized a significantly greater number of CD34+ cells compared to G-CSF plus placebo [13]. While this is promising, accumulating data suggests that MM therapies such as daratumumab [14] or lenalidomide [15] may negatively impact HSC mobilization. Mobilization failure can lead to potential loss of ASCT as a treatment option or significant toxicity from repeated mobilization attempts [16].
Methods
Cell culture
MDA-MB-231, Namalwa and MM.1S cells were purchased from the American Type Culture Collection (ATCC) and U937 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured in RPMI 1640 with ATCC modification (Gibco, A1049101), supplemented with 10% fetal bovine serum (FBS) and 100 U/ml of Penicillin–Streptomycin.
Inhibition of CXCL12 binding to CXCR4
Competitive binding assays were performed using the TagLite® system (CisBio). After CXCR4 terbium-labeled cells were plated, 5 μL of GPC-100 or AMD3100 were added, followed by 5 μL of 20 nM red fluorescently labeled CXCL12. After a 3-h incubation at room temperature, the plate was read on an EnVision plate reader (PerkinElmer). Inhibitory constants (Ki) for the respective compounds were determined by plotting the normalized HTRF ratio versus the compound concentrations using non-linear regression competitive binding “one site–fit Ki equation.” The HTRF ratio was calculated as the emission ratio of 665 nm/620 nm proportional to the amount of CXCL12 bound to CXCR4.
Results
GPC-100 shows distinct binding modes and higher binding affinity for CXCR4 compared to AMD3100
The chemical composition of GPC-100 demonstrates distinct divergence from AMD3100 (Fig 1a). To understand these differences at a molecular level, we use in silico docking experiments of the two antagonists to an inactive structure of CXCR4. Previous studies with various CXCR4 antagonists have identified major and minor pockets of interactions within the CXCR4 orthosteric binding site [43]. The induced-fit algorithm from Schrödinger was used for GPC-100 [43, 44]. Previously validated residues with secondary amines, namely Asp97 and Asp262 [50], contributed to interactions of GPC-100 and AMD3100 with CXCR4. However, the phosphoryl group of GPC-100 that is absent in AMD3100 makes an ionic interaction with Arg188, and hydrogen bond with Gln200 (Fig 1b). These energetically favorable interactions suggest enhanced anchor points for GPC-100 that are absent in AMD3100.
Discussion
In the present study, we provide in vitro and in vivo characterization of GPC-100, a novel CXCR4 antagonist and hematopoietic cell mobilizer. We also demonstrate the functional interactions between CXCR4 and β2AR, which support the addition of propranolol to GPC-100 as a novel strategy for HSC mobilization. GPC-100 demonstrated efficacy in disrupting CXCL12-mediated calcium signaling, cell migration and β-arrestin recruitment to CXCR4 that was comparable with AMD3100. However, GPC-100 was a better mobilizer in vivo. The competitive binding assay revealed that GPC-100 possessed superior binding to CXCR4 compared to AMD3100. We speculate that this could be due to the chemical scaffold of GPC-100 forming enhanced interactions in the CXCR4 binding pocket stabilizing the receptor differently from AMD3100. The functional consequences of ligand binding, whether activating or inhibiting, are dependent on the structural characteristics of the ligand-receptor complex [67, 68].
Acknowledgments
We thank Glenn Cantor and Madeleine Héroux, consultants for GPCR Therapeutics, for providing editing and review support during preparation of this manuscript.
Citation: Sukhtankar DD, Fung JJ, Kim M-n, Cayton T, Chiou V, Caculitan NG, et al. (2023) GPC-100, a novel CXCR4 antagonist, improves in vivo hematopoietic cell mobilization when combined with propranolol. PLoS ONE 18(10): e0287863. https://doi.org/10.1371/journal.pone.0287863
Editor: Nicolas N. Nassar, Cincinnati Children’s Hospital Medical Center, UNITED STATES
Received: June 12, 2023; Accepted: September 27, 2023; Published: October 25, 2023
Copyright: © 2023 Sukhtankar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The data underlying the results presented in the study are available from https://www.ebi.ac.uk/biostudies/studies/S-BSST1177.
Funding: This work was mainly supported by GPCR Therapeutics, Inc and in part by the National Research Foundation of Korea (2020R1A5A1018081). GPCR Therapeutics provided support in the form of salaries to all listed authors, who are currently or formerly employed by GPCR Therapeutics. National Research Foundation of Korea provided support in the form of research grant for WKH. Funders did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
Competing interests: DDS, TC, VC, NGC, YJ, SK, JML, JC, SM, AC, JYL, GK, JYJ, DSS, and PMC are current employees. JJF, MK, PZ, SJK, YDJ and EHK were previously employed by GPCR Therapeutics, Inc. DDS and WKH are co-founders of GPCR Therapeutics. DSS, JJF, NGC, EHK, PZ and PMC have a patent pending for uses of GPCR inhibitors. The commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0287863#abstract0
