L-CCL2 aptamer

•



Timeline

2007

The aptamer was isolated for the first time[1]

2008

The physical and chemical properties of NOX-E36 RNA aptamer[2]

2008

Ccl2 blockade with mNOX-E36–3′PEG effectively blocked macrophage recruitment into the glomerular and interstitial compartments of the kidney[3]

2009

Ccl2 mediates the recruitment of glomerular and interstitial macrophages but this mechanism does not contribute to the progression of Alport nephropathy in Col4a3-deficient mice[4]

2012

Pharmacological inhibition of MCP-1 with mNOX-E36 was investigated[5]

2014

Inhibition of monocyte infiltration by targeting the chemokine CCL2 prevented fibrosis-associated angiogenesis, but did not halt fibrosis progression[6]

2014

Pharmacological inhibition of Ly-6C(+) monocyte recruitment using the CCL2-inhibitor mNOX-E36 accelerated regression from toxic and metabolic liver fibrosis[7]

2015

The crystal structure of the aptamer have been deterimined[8]

2015

Spiegelmer-based inhibition of MCP-1/CCR2 with the compound mNOX-E36 resulted in moderate but statistically significant improvements in graft survival[9]

2017

Inhibition of the CCL2/CCL2 receptor axis with NOX-E36 was generally safe and well-tolerated[10]

2017

Inhibition of CCL2 in diabetic nephropathy effectively reduced proteinuria and was associated with restoration of glomerular endothelial glycocalyx size[11]

Description

In 2007, Anders, H. J. isolated the L-enantiomeric RNA oligonucleotide mNOX-E36, a so-called Spiegelmer that binds murine CCL2 with high affinity and neutralizes its action in vitro and in vivo. In 2015, Betzel C et al. chemically synthesized the NOX-E36 L-aptamer, a Spiegelmer designed to bind to L-CCL2. To elucidate its structure, they employed single-wavelength anomalous diffraction (SAD) and refined the structure to a resolution of 2.05 Å. This technique allowed them to gain detailed insights into the interactions between the L-aptamer and its target protein. The resulting structural data provided a comprehensive understanding of the binding interface and the conformational details of the L-aptamer. This high-resolution structure was crucial for understanding the molecular basis of the L-aptamer's high affinity and specificity for L-CCL2. Such detailed structural information is essential for optimizing the design of Spiegelmers as potential therapeutic agents[1,8].



SELEX

In 2007, Anders, H. J. et al. generated the Spiegelmer mNOX-E36 through a rigorous in vitro selection process. A DNA library with 40 internal random positions was used to facilitate the identification of high-affinity Spiegelmers. The library underwent 11 rounds of selection, which allowed for the systematic enrichment of RNA sequences with high binding affinity for murine D-CCL2. Following these selection rounds, the most prevalent and effective RNA sequence was identified. This sequence was then truncated to obtain the final candidate, mNOX-E36. The selection process was crucial in ensuring that mNOX-E36 possessed the desired properties for therapeutic applications, particularly in the context of lupus nephritis. The successful identification of mNOX-E36 highlights the potential of Spiegelmers as a novel class of therapeutic agents[1].

Detailed information are accessible on SELEX page.



Structure

2D representation

In 2007, Anders, H. J. et al. employed an in vitro selection process to generate the Spiegelmer mNOX-E36. mNOX-E36 is a 45-nucleotide and mouse-CCL2-specific Spiegelmer aptamer. NOX-E36 L-aptamer is a 40-nucleotide and Human-CCL2-specific Spiegelmer aptamer. Here we use ribodraw to complete the figure, through the 3D structure information. The NOX-E36 L-aptamer was named by Anders, H. J. et al. in the article[1].

5'-GCACGUCCCUCACCGGUGCAAGUGAAGCCGAGGCUCUGCG-3'

drawing

3D visualisation

NOX-E36 L-aptamer is a Spiegelmer designed to bind to L-CCL2. To elucidate its crsytal structure, they employed single-wavelength anomalous diffraction (SAD) and refined the structure to a resolution of 2.05 Å. This technique allowed them to gain detailed insights into the interactions between the L-aptamer and its target protein. The resulting structural data provided a comprehensive understanding of the binding interface and the conformational details of the L-aptamer. The PDB ID of this structure is 4R8I[8].

Additional available structures that have been solved and detailed information are accessible on Structures page.

(Clicking the "Settings/Controls info" to turn Spin off)      

drawing PDBe Molstar




Binding pocket

Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 4R8I. L-CCL2 (shown in vacuumm electrostatics), blue is positive charge, red is negative charge. Right: The hydrogen bonds of binding sites of the aptamer bound with L-CCL2.

drawing drawing


Ligand information

SELEX ligand

Betzel, C. et al. investigated the ion dependence of L-aptamer binding using surface plasmon resonance (SPR), revealing a strong dependence on Ca2+, which appears to be important for proper folding of the L-aptamer and will likely occupy the position of Sr2+ observed in the structure. And they also studied the effect of the mutations on L-aptamer affinity and measured the L-aptamer’s affinity using SPR[8].

Name Sequence Ligand Affinity
L-CCL2 aptamer 5'-GCACGUCCCUCACCGGUGCAAGUGAAGCCGAGGCUCUGCG-3' L-CCL2 1.40 ± 0.16 nM

Structure ligand

Acts as a ligand for C-C chemokine receptor CCR2. Signals through binding and activation of CCR2 and induces a strong chemotactic response and mobilization of intracellular calcium ions. Exhibits a chemotactic activity for monocytes and basophils but not neutrophils or eosinophils. May be involved in the recruitment of monocytes into the arterial wall during the disease process of atherosclerosis.-----From Pfam

UniProt ID: uniquely identifies protein sequences in the UniProt database, a resource for protein information.

Pfam: a widely recognised database of protein families and domains.

GenBank: maintained by NCBI(National Center for Biotechnology Information), is a database of nucleotide sequences from various organisms, vital for genetic and molecular biology research.

Mass: an intrinsic property of a body.

Uniprot ID Pfam Mass Protein sequence PDB ID GenBank
P13500 P13500 8.82 kDa MQPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT 1DOK 9606
drawing

Similar compound

We used the Dail server website to compare the structural similarities of ligand proteins, and chose the top 10 in terms of similarity for presentation.

Dail server website: a network service for comparing protein structures in 3D. Dali compares them against those in the Protein Data Bank (PDB).

Z-score: a standard score that is converted from an original score. The list of neighbours is sorted by Z-score. Similarities with a Z-score lower than 2 are spurious.

RMSD: (Root Mean Square Deviation) is used to measure the degree to which atoms deviate from the alignment position.

PDB: PDB ID+ chain name.

PDB Z-score RMSD Description
1DOK-A 15.7 0 Monocyte chemoattractant protein 1
7SO0-B 12 0.7 Evasin p974
2VXW-A 12 1.5 C-C motif chemokine 5
1NR4-E 11.3 1.5 Thymus and activation-regulated chemokine
5EKI-E 10.4 1.5 C-C motif chemokine 21
2JYO-A 9.4 2.1 C-C motif chemokine 20 (small-inducible cytokine)
1B2T-A 8.7 3.5 Protein (fractalkine)
2MP1-A 8.3 2.0 C-C motif chemokine 19
1O80-A 7.9 2.4 Small inducible cytokine b10
1F9P-A 7.4 1.6 Connective tissue activating peptide-iii


References

[1] Spiegelmer inhibition of CCL2/MCP-1 ameliorates lupus nephritis in MRL-(Fas)lpr mice.
Kulkarni, O., Pawar, R. D., Purschke, W., Eulberg, D., Selve, N., Buchner, K., Ninichuk, V., Segerer, S., Vielhauer, V., Klussmann, S., & Anders, H. J.
Journal of the American Society of Nephrology : JASN, 18(8), 2350–2358. (2007)
[2] Physicochemical stability of NOX-E36, a 40mer L-RNA (Spiegelmer) for therapeutic applications.
Maasch, C. , Buchner, K. , Eulberg, D. , Vonhoff, S. , & Klussmann, S.
Nucleic acids symposium series, (52), 61–62. (2008)
[3] Late onset of Ccl2 blockade with the Spiegelmer mNOX-E36-3'PEG prevents glomerulosclerosis and improves glomerular filtration rate in db/db mice.
Ninichuk, V. , Clauss, S. , Kulkarni, O. , Schmid, H. , Segerer, S. , Radomska, E. , Eulberg, D. , Buchner, K. , Selve, N. , Klussmann, S. , & Anders, H. J
The American journal of pathology, 172(3), 628–637. (2008)
[4] Ccl2/Mcp-1 blockade reduces glomerular and interstitial macrophages but does not ameliorate renal pathology in collagen4A3-deficient mice with autosomal recessive Alport nephropathy.
Clauss, S. , Gross, O. , Kulkarni, O. , Avila-Ferrufino, A. , Radomska, E. , Segerer, S. , Eulberg, D. , Klussmann, S. , & Anders, H. J.
The Journal of pathology, 218(1), 40–47. (2009)
[5] Pharmacological inhibition of the chemokine CCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury.
Baeck, C. , Wehr, A. , Karlmark, K. R. , Heymann, F. , Vucur, M. , Gassler, N. , Huss, S. , Klussmann, S. , Eulberg, D. , Luedde, T. , Trautwein, C. , & Tacke, F.
Gut,61(3), 416–426. (2012)
[6] CCL2-dependent infiltrating macrophages promote angiogenesis in progressive liver fibrosis.
Ehling, J. , Bartneck, M. , Wei, X. , Gremse, F. , Fech, V. , Möckel, D. , Baeck, C. , Hittatiya, K. , Eulberg, D. , Luedde, T. , Kiessling, F. , Trautwein, C. , Lammers, T. , & Tacke, F.
Gut,63(12), 1960–1971. (2014)
[7] Pharmacological inhibition of the chemokine C-C motif chemokine ligand 2 (monocyte chemoattractant protein 1) accelerates liver fibrosis regression by suppressing Ly-6C(+) macrophage infiltration in mice.
Baeck, C. , Wei, X. , Bartneck, M. , Fech, V. , Heymann, F. , Gassler, N. , Hittatiya, K. , Eulberg, D. , Luedde, T. , Trautwein, C. , & Tacke, F.
Hepatology (Baltimore, Md.), 59(3), 1060–1072. (2014)
[8] Crystal structure of a mirror-image L-RNA aptamer (Spiegelmer) in complex with the natural L-protein target CCL2.
Oberthür D, Achenbach J, Gabdulkhakov A, Buchner K, Maasch C, Falke S, Rehders D, Klussmann S, Betzel C.
Nat Commun, 6:6923 (2015)
[9] Spiegelmer Inhibition of MCP-1/CCR2--Potential as an Adjunct Immunosuppressive Therapy in Transplantation.
Kalnins, A. , Thomas, M. N. , Andrassy, M. , Müller, S. , Wagner, A. , Pratschke, S. , Rentsch, M. , Klussmann, S. , Kauke, T. , Angele, M. K. , Bazhin, A. V. , Fischereder, M. , Werner, J. , Guba, M. , & Andrassy, J.
Scandinavian journal of immunology, 82(2), 102–109. (2015)
[10] C-C motif-ligand 2 inhibition with emapticap pegol (NOX-E36) in type 2 diabetic patients with albuminuria.
Menne, J., Eulberg, D., Beyer, D., Baumann, M., Saudek, F., Valkusz, Z., Więcek, A., Haller, H., & Emapticap Study Group
Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association,  32(2), 307–315. (2017)
[11] Systemic Monocyte Chemotactic Protein-1 Inhibition Modifies Renal Macrophages and Restores Glomerular Endothelial Glycocalyx and Barrier Function in Diabetic Nephropathy.
Boels, M. G. S. , Koudijs, A. , Avramut, M. C. , Sol, W. M. P. J. , Wang, G. , van Oeveren-Rietdijk, A. M. , van Zonneveld, A. J. , de Boer, H. C. , van der Vlag, J. , van Kooten, C. , Eulberg, D. , van den Berg, B. M. , IJpelaar, D. H. T. , & Rabelink, T. J.
The American journal of pathology, 187(11), 2430–2440. (2017)