Computational modeling and experimental validation of the EPI-X4/CXCR4 complex allows rational design of small peptide antagonists

AbstractEPI-X4, a 16-mer fragment of albumin, is a specific endogenous antagonist and inverse agonist of the CXC-motif-chemokine receptor 4 (CXCR4) and thus a key regulator of CXCR4 function. Accordingly, activity-optimized synthetic derivatives of EPI-X4 are promising leads for the therapy of CXCR4-linked disorders such as cancer or inflammatory diseases. We investigated the binding of EPI-X4 to CXCR4, which so far remained unclear, by means of biomolecular simulations combined with experimental mutagenesis and activity studies. We found that EPI-X4 interacts through its N-terminal residues with CXCR4 and identified its key interaction motifs, explaining receptor antagonization. Using this model, we developed shortened EPI-X4 derivatives (7-mers) with optimized receptor antagonizing properties as new leads for the development of CXCR4 inhibitors. Our work reveals the molecular details and mechanism by which the first endogenous peptide antagonist of CXCR4 interacts with its receptor and provides a foundation for the rational design of improved EPI-X4 derivatives.

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Rational Design of Conformationally Constrained Cyclopentapeptide Antagonists for C-X-C Chemokine Receptor 4 (CXCR4)

Journal of Medicinal Chemistry ◽

10.1021/jm300926y ◽

2012 ◽

Vol 55 (22) ◽

pp. 10287-10291 ◽

Cited By ~ 23

Author(s):

Jignesh Mungalpara ◽

Stefanie Thiele ◽

Øystein Eriksen ◽

Johann Eksteen ◽

Mette M. Rosenkilde ◽

...

Keyword(s):

Chemokine Receptor ◽

Rational Design ◽

Conformationally Constrained ◽

Chemokine Receptor 4

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Biomolecular models of EPI-X4 binding to CXCR4 allow the rational optimization of peptides with therapeutic potential

10.1101/2020.10.23.352708 ◽

2020 ◽

Author(s):

Pandian Sokkar ◽

Mirja Harms ◽

Christina Stürzel ◽

Andrea Gilg ◽

Gönül Kizilsavas ◽

...

Keyword(s):

Therapeutic Potential ◽

Inflammatory Diseases ◽

Rational Drug Design ◽

Downstream Signaling ◽

Cxcr4 Signaling ◽

Binding Behavior ◽

Rational Drug ◽

Chemokine Ligand ◽

Biomolecular Simulations ◽

Chemokine Receptor 4

ABSTRACTThe Endogenous Peptide Inhibitor of CXCR4 (EPI-X4) is a body-own fragment of albumin and specific antagonist of the CXC-motif-chemokine receptor 4 (CXCR4). CXCR4 signaling is induced by its sole chemokine ligand CXCL12 and is involved in a plethora of functions including cell homing, differentiation, survival and angiogenesis. Consequently, dysregulation of CXCR4 is involved in a variety of disorders, such as cancer or inflammatory diseases, making CXCR4 an attractive drug target. EPI-X4 and derivatives with increased CXCR4 binding affinities represent promising leads as CXCR4 antagonists and have shown therapeutic activity in mouse models of inflammatory diseases. However, it is currently unclear how EPI-X4 and its derivatives interact with CXCR4. Here, by combining biomolecular simulations with experimental mutagenesis and activity studies we investigated the binding behavior of EPI-X4 to CXCR4 at the molecular level. Our work allowed us to show that the EPI-X4 peptide interacts primarily in the minor pocket of CXCR4 through its N-terminal residues. The biomolecular interactions highlighted by the computational studies are in good agreement with the experimental mutagenesis data. Moreover, we found that the N-terminal seven amino-acids of EPI-X4 (a 16-mer) and its improved derivatives (12-mers) are sufficient for CXCR4 binding, which led to the development of shorter leads with optimized CXCR4 antagonizing properties. Collectively, we here established how EPI-X4 binds to its receptor and used this knowledge for rational drug design. The new peptide variants developed by us are more potent in terms of inhibiting CXCR4-downstream signaling and cancer cell migration, without toxic effects.

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Stoichiometry and geometry of the CXC chemokine receptor 4 complex with CXC ligand 12: Molecular modeling and experimental validation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1417037111 ◽

2014 ◽

Vol 111 (50) ◽

pp. E5363-E5372 ◽

Cited By ~ 52

Author(s):

Irina Kufareva ◽

Bryan S. Stephens ◽

Lauren G. Holden ◽

Ling Qin ◽

Chunxia Zhao ◽

...

Keyword(s):

Molecular Modeling ◽

Chemokine Receptor ◽

Experimental Validation ◽

Cxc Chemokine Receptor 4 ◽

Cxc Chemokine ◽

Chemokine Receptor 4

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Synthetic derivatives of pulchrol: An antiparasitic natural product

Planta Medica ◽

10.1055/s-0036-1596772 ◽

2016 ◽

Vol 81 (S 01) ◽

pp. S1-S381

Author(s):

P Terrazas ◽

O Sterner

Keyword(s):

Natural Product ◽

Synthetic Derivatives ◽

Derivatives Of

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Faculty Opinions recommendation of A chemokine receptor CXCR2 macromolecular complex regulates neutrophil functions in inflammatory diseases.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13928956.15385056 ◽

2012 ◽

Author(s):

Richard L Stevens

Keyword(s):

Chemokine Receptor ◽

Inflammatory Diseases ◽

Macromolecular Complex ◽

Chemokine Receptor Cxcr2

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Synthesis, Cytotoxicity and Antioxidant Activity of New Analogs of RC-121 Synthetic Derivatives of Somatostatin

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520618666180417164344 ◽

2019 ◽

Vol 18 (10) ◽

pp. 1417-1424 ◽

Cited By ~ 2

Author(s):

Emilia Naydenova ◽

Diana Wesselinova ◽

Svetlana Staykova ◽

Ivan Goshev ◽

Ljubomir Vezenkov

Keyword(s):

Antioxidant Activity ◽

Antioxidant Capacity ◽

Cell Line ◽

Cancer Cell ◽

Cancer Cell Line ◽

Colorectal Cancer Cell Line ◽

Cervical Cancer Cell Line ◽

Synthetic Derivatives ◽

Derivatives Of

Background: Based on the structure of RC-121 (D-Phe-c (Cys-Tyr-D-Trp-Lys-Val-Cys)-Thr-NH2, - synthetic derivatives of somatostatin), some analogs were synthesized and tested for in vitro cytotoxic and antioxidant activity. Objectives: The new analogs were modifyed at position 5 with Dap (diaminopropanoic acid), Dab (diaminobutanoic acid) and Orn and at position 6 with the unnatural amino acids Tle (t-leucine). Methods: The in vitro cytotoxic effects of the substances were investigated against a panel of human tumor cell lines HT-29 (Human Colorectal Cancer Cell Line), MDA-MB-23 (Human Breast Cancer Cell Line), Hep G-2 (Human Hepatocellular Carcinoma Cell Line) and HeLa (cervical cancer cell line). The antioxidant capacities were tested by ORAC (Oxygen Radical Antioxidant Capacity) and HORAC (Hydroxyl Radical Averting Capacity) methods. Results: All substances expressed significantly higher antioxidant capacity by comparison with galic acid and Trolox. All substances showed considerable antioxidant capacity as well. Compound 2T (D-Phe-c(Cys-Tyr-DTrp- Dap-Tle-Cys)-Thr-NH2)had the highest antioxidant effect. The compound 4T (D-Phe-c(Cys-Tyr-D-Trp- Orn-Tle-Cys)-Thr-NH2) displayed antiproliferative effect on HeLa cells with IC50 30 µM. The peptide analog 3T (D-Phe-c(Cys-Tyr-D-Trp-Lys-Tle-Cys)-Thr-NH2) exerted the most pronounced inhibition on the cell vitality up to 53%, 56% and 65% resp. against MDA-MB-23, Hep G-2, HeLa in the higher tested concentration. Conclusion: The somatostatin analogs showed moderate influence on the vitality of different tumor cells and could be used in changing their pathology.

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Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA

Scientific Reports ◽

10.1038/s41598-021-81743-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Peter D. Leitner ◽

Ilja Vietor ◽

Lukas A. Huber ◽

Taras Valovka

Keyword(s):

Dna Binding ◽

Rational Design ◽

Dissociation Constants ◽

Inflammatory Diseases ◽

Dimethyl Fumarate ◽

Bioinformatic Analysis ◽

Quantitative Detection ◽

Regulatory Sequences ◽

Wide Range ◽

Inhibitory Drug

AbstractThe nuclear factor kappa B (NF-κB) family of dimeric transcription factors regulates a wide range of genes by binding to their specific DNA regulatory sequences. NF-κB is an important therapeutic target linked to a number of cancers as well as autoimmune and inflammatory diseases. Therefore, effective high-throughput methods for the detection of NF-κB DNA binding are essential for studying its transcriptional activity and for inhibitory drug screening. We describe here a novel fluorescence-based assay for quantitative detection of κB consensus double-stranded (ds) DNA binding by measuring the thermal stability of the NF-κB proteins. Specifically, DNA binding proficient NF-κB probes, consisting of the N-terminal p65/RelA (aa 1–306) and p50 (aa 1–367) regions, were designed using bioinformatic analysis of protein hydrophobicity, folding and sequence similarities. By measuring the SYPRO Orange fluorescence during thermal denaturation of the probes, we detected and quantified a shift in the melting temperatures (ΔTm) of p65/RelA and p50 produced by the dsDNA binding. The increase in Tm was proportional to the concentration of dsDNA with apparent dissociation constants (KD) of 2.228 × 10–6 M and 0.794 × 10–6 M, respectively. The use of withaferin A (WFA), dimethyl fumarate (DMF) and p-xyleneselenocyanate (p-XSC) verified the suitability of this assay for measuring dose-dependent antagonistic effects on DNA binding. In addition, the assay can be used to analyse the direct binding of inhibitors and their effects on structural stability of the protein probe. This may facilitate the identification and rational design of new drug candidates interfering with NF-κB functions.

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