Allosteric modulation of the CXCR4:CXCL12 axis by targeting receptor nanoclustering via the TMV-TMVI domain

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 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.

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.