Allosteric modulation of the CXCR4:CXCL12 axis by targeting receptor nanoclustering via the TMV-TMVI domain
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eLife assessment
This important study identifies novel small molecule antagonists of CXCR4 that disrupt nanocluster formation and chemotactic function without blocking CXCL12 binding and downstream signals. The conclusions are based on solid evidence, but the work could be improved by including kinetic and dose information on the most active inhibitors. We also note that modeling and mutagenesis implicate helix V and VI in an allosteric mechanism, but that the description of the modeling is not sufficiently detailed such that others could replicate it.
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Abstract
CXCR4 is a ubiquitously expressed chemokine receptor that regulates leukocyte trafficking and arrest in homeostatic and pathological states, and also participates in organogenesis, HIV-1 infection and tumor development. Despite the potential therapeutic benefit of CXCR4 antagonists, so far only one, plerixafor (AMD3100), which blocks the ligand-binding site, has reached the clinic. Recent advances in imaging and biophysical techniques have provided a richer understanding of the membrane organization and dynamics of this receptor. CXCL12 activation of CXCR4 reduces the number of CXCR4 monomers/dimers at the cell membrane and increases the formation of large nanoclusters, which are largely immobile and are required for correct cell orientation towards chemoattractant gradients. Mechanistically, CXCR4 activation involves a structural motif defined by residues on TMV and TMVI of CXCR4. Using this structural motif as a template, we performed in silico molecular modeling followed by in vitro screening of a small compound library to search for allosteric antagonists of CXCR4 that do not affect CXCL12 binding. We identified AGR1.137, a small compound that abolishes CXCL12-mediated receptor nanoclustering and dynamics and blocks the ability of cells to sense CXCL12 gradients both in vitro and in vivo without altering ligand binding or receptor internalization. CXCR4 is a ubiquitous chemokine receptor that regulates leukocyte trafficking and arrest in homeostatic and pathological states. Yet, the only commercial CXCR4 antagonist approved for clinical use is plerixafor (AMD3100), a small compound that blocks the ligand-binding site. Unfortunately, its clinical application is limited by poor pharmacokinetics and adverse effects associated with long-term administration. Here, we performed in silico analyses of a small aromatic compound library followed by in vitro screening to identify allosteric CXCR4 antagonists that abrogate the ability of cells to sense chemoattractant gradients without altering other ligand-mediated functions such as blockade of cAMP production or receptor internalization. The selected compounds also acted in vivo, as demonstrated by reduced tumorigenesis and metastasis in a zebrafish tumor model. Our study describes a new approach to selectively alter some GPCR functions without the need for abolishing all receptor functionality.
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Author Response
Many thanks for handling our manuscript (eLife-RP-RA-2023-93968) entitled "Allosteric modulation of the CXCR4:CXCL12 axis by targeting receptor nanoclustering via the TMV-TMVI domain", by García-Cuesta et al. We are delighted to hear your willingness to consider our manuscript following appropriate revision. We have carefully read the referees' commentaries and have organized new experiments to address their specific queries.
Reviewer #1 (Public Review)
The computational methodology is going to be carefully reviewed. In particular to justify the software and techniques used in this manuscript. We will also describe the method for identifying the pocket on the CXCR4 structure as well as the workflow used to explain the transition from docking evaluation to MD analyses. Additionally, we will conduct experiments to enhance …
Author Response
Many thanks for handling our manuscript (eLife-RP-RA-2023-93968) entitled "Allosteric modulation of the CXCR4:CXCL12 axis by targeting receptor nanoclustering via the TMV-TMVI domain", by García-Cuesta et al. We are delighted to hear your willingness to consider our manuscript following appropriate revision. We have carefully read the referees' commentaries and have organized new experiments to address their specific queries.
Reviewer #1 (Public Review)
The computational methodology is going to be carefully reviewed. In particular to justify the software and techniques used in this manuscript. We will also describe the method for identifying the pocket on the CXCR4 structure as well as the workflow used to explain the transition from docking evaluation to MD analyses. Additionally, we will conduct experiments to enhance the results and address the specific feedback provided, ultimately improving the overall reliability.
Reviewer #2 (Public Review)
Although the paper was initiated by titrating the compounds in migration experiments, we are going to add new kinetics and titration of concentrations in these experiments. In addition, we are going to change the way in which we present the data from the singlemolecule tracking experiments. We will add a representative video of each experimental condition, and include some of the mean square displacement curves to support our data on the analysis of the diffusion coefficient (D1-4) to give a more conclusive view of receptor clustering. Regarding the tumorigenesis experiments we will include the individual data points and we will try to perform kinetics with distinct concentrations of the drug.
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eLife assessment
This important study identifies novel small molecule antagonists of CXCR4 that disrupt nanocluster formation and chemotactic function without blocking CXCL12 binding and downstream signals. The conclusions are based on solid evidence, but the work could be improved by including kinetic and dose information on the most active inhibitors. We also note that modeling and mutagenesis implicate helix V and VI in an allosteric mechanism, but that the description of the modeling is not sufficiently detailed such that others could replicate it.
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Reviewer #1 (Public Review):
Summary:
The manuscript, titled "Allosteric Modulation of the CXCR4:CXCL12 Axis by Targeting Receptor Nanoclustering via the TMV-TMVI Domain," presents a compelling investigation into the development of a potential anti-cancer therapeutic agent. The study focuses on targeting specific CXCR4 intermolecular interactions via an allosteric antagonist which binds proximal to the orthosteric ligand binding site. The novel compounds developed aim to mitigate tumor dissemination, proliferation, and metastasis in transgenic Zebrafish models implanted with HeLa cells.Strengths:
The study holds significant promise, offering a novel approach to addressing the targeted modulation of CXCR4. The multidisciplinary methodology employed is commendable, providing a comprehensive understanding of the underlying molecular …Reviewer #1 (Public Review):
Summary:
The manuscript, titled "Allosteric Modulation of the CXCR4:CXCL12 Axis by Targeting Receptor Nanoclustering via the TMV-TMVI Domain," presents a compelling investigation into the development of a potential anti-cancer therapeutic agent. The study focuses on targeting specific CXCR4 intermolecular interactions via an allosteric antagonist which binds proximal to the orthosteric ligand binding site. The novel compounds developed aim to mitigate tumor dissemination, proliferation, and metastasis in transgenic Zebrafish models implanted with HeLa cells.Strengths:
The study holds significant promise, offering a novel approach to addressing the targeted modulation of CXCR4. The multidisciplinary methodology employed is commendable, providing a comprehensive understanding of the underlying molecular interactions. The proposed workflow, although requiring some adjustments, is reasonable and has the potential to make a substantial impact in the field.Weaknesses:
Despite the brilliance of the concept and its potential impact, the computational approach appears somewhat superficial and lacks essential considerations. A comprehensive revision of the computational methodology is strongly recommended, with a focus on addressing key points. Additionally, the experimental section should be modified accordingly to align with the refined results. While the study's foundations are promising, its current state warrants a thorough revision to enhance its scientific rigor and overall robustness. -
Reviewer #2 (Public Review):
Summary:
This work describes a new pharmacological targeting approach to inhibit selective functions of the ubiquitously expressed chemokine receptor CXCR4, a potential target of immunomodulatory or anti-cancer treatments. Overall, the results build a strong case for the potential of this new compound to target specific functions of CXCR4, particularly linked to tumorigenesis. However, a more thorough evaluation of the function of the compound as well as future studies in mammalian model systems are needed to better assess the promise of the compound.Strengths:
The work elegantly utilizes in silico drug modelling to propose new small molecule compounds with specific features. This way, the authors designed compound AGR1.137, which abolishes ligand-induced CXCR4 receptor nanoclustering and the subsequent …Reviewer #2 (Public Review):
Summary:
This work describes a new pharmacological targeting approach to inhibit selective functions of the ubiquitously expressed chemokine receptor CXCR4, a potential target of immunomodulatory or anti-cancer treatments. Overall, the results build a strong case for the potential of this new compound to target specific functions of CXCR4, particularly linked to tumorigenesis. However, a more thorough evaluation of the function of the compound as well as future studies in mammalian model systems are needed to better assess the promise of the compound.Strengths:
The work elegantly utilizes in silico drug modelling to propose new small molecule compounds with specific features. This way, the authors designed compound AGR1.137, which abolishes ligand-induced CXCR4 receptor nanoclustering and the subsequent directed cell migration without affecting ligand binding itself or some other ligand-induced signaling pathways. The authors have used a relatively broad set of experiments to validate and demonstrate the effects of the drug. Importantly, the authors also test AGR1.137 in vivo, using a zebrafish model of tumorigenesis and metastasis. A relatively strong inhibitory effect of the compound is reported.Weaknesses:
The data would be significantly strengthened by adding kinetics and titration of concentrations. This is particularly important as it is the first description of these particular compounds and would help to evaluate the potency and possible side effects of the drug.The authors carry out single-molecule tracking experiments to analyze nanoclustering of CXCR4 upon ligand binding. This complex data is presented in a sub-optimal manner. Representative images of the data should be included together with more thorough analysis tools like autocorrelation function or mean square displacement to get a more conclusive view of receptor clustering and the effects of the compound.
In the in vivo tumorigenesis experiments, again more kinetics and different concentrations of the drug would generate more convincing data. Also, the individual data points should be visualized to allow full evaluation of the data, throughout the experiments.
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