N-Methyl Mesoporphyrin IX Bound to Human Telomeric G-Quadruplex DNA

Mason McCool '17, Cole Meier '19


I. Introduction

Guanine rich DNA sequences have the ability to form tertiary structures, called G-quadruplexes (GQs). Their related consensus sequences are found in various organisms, greatly conserved in mammals, but present less frequenently in lower organism such as bacteria.1 GQs are four stranded DNA sequences, differing from the normal duplex DNA structure, and can form inter or intra molecularly. These structures can form at various parts of the human genome, such as gene promoters, replication origins, and intron/exon borders. However, GQs are most prevalently found within telomeres, where there are up to 10,000 bases of TTAGGG repeats, that are able to readily form these structures. They form at the 3' overhang, which is a single stranded portion allowing for the formation of intramolecular GQs, dGGG(TTAGGG) 3.2

One major importance of 3' overhang GQs in-vivo can be attributed to their end capping that protects chromosome ends. Because of their extensive four stranded bulky structure, they provide resistance against nucleases. Along these same lines, it also has the ability to inhibit telomerase (the enzyme that adds nucleotides to extend the telomere when it is shortened due to DNA replication cycles), thus regulating it's activity and relative telomere length. This aspect of GQs has been taken advantage as a target for anti-cancer drug therapy.3 There are ligands that can bind and stabilize GQs, leading to greater telomerase4 inhibition. If a significant amount of inhibition occurs, this will eventually lead to telomere shortening and eventually cell senescence or apoptosis.3

There have only been a moderate amount of these structures determined for different human telomere GQ-ligand complexes. Many of these are low resolution, so specific features of ligand-GQ interactions cannot be determined. It is also noteworth that no highly selective GQ ligands have been studied, until the study of N-methyl mesoporphyrin IX (). Porphyrins contain a conjugated macrocyclic structure and can have various substituents. NMM most notably has two carboxylate groups on the exterior portion of the molecule and an N-methyl group that protrudes down away from the aromatic plane in the center of the molecule.6

NMM was originally studied as an inhibitor of ferrochelatase, the enzyme that catalyzes the insertion of iron for heme biosynthesis.7 However, now it is of interest because of its selectivity for parallel GQs unlike other anti-cancer GQ ligands studied previously.

II. G-Quadruplex Structure

GQs structures form from guanines held together by Hoogsteen base-pairing between four guanines forming a tetrad structure. There is hydrogen bonding between as well as of two separate guanines, forming 8 hydrogen bonds total per .

Then the three tetrads stack upon each other due to stabilizing between the aromatic groups on guanine, similar to the alpha helical base stacking that stabilizes the general DNA secondary structure. Three guanines that occur in sequence are stacked upon each other. Each of the guanine sequences are connected through three base loops (TTA), occuring three times to connect all four guanine sequences and forming three tetrads . These structures are further stabilized in physiological buffer conditions by monovalent cations such as Na+ and K+ that interact with the carbonyl oxygen on the guanines. There are 3 cations present per GQ structure, one for each tetrad, that stabilize via charge screening.5

While the general GQ structure is outlined by guanine base pairing and cation interactions, there can be different alignments of the sequence or sequences of nucleotides that result in a specific type of GQ structure. When all three guanine base regions are oriented in the same directionality (upward/downward relatively) it is considered parallel, while if they are not it is antiparallel.5 As mentioned prior, NMM prefers the parrallel conformation of GQs . It can even induce a conformational change to the parallel GQ structure, unique compared to other ligands. Knowing the structure of GQs alone, helps determine possible ligands that can be used to further stabilize the GQ and be used as anti-cancer drugs.

III. G-Quadruplex Dimer Formation, Not Indicative of Native State

When in a crystal structure the telomeric DNA GQs form in dimers. The 5’ face of both of the GQs form a dimer through pi stacking interactions and a bridging K+ ion. In addition, there is reverse base pairing between A1 of one DNA GQ strand and T12 of the other DNA GQ strand .5 There can even be some interactions between multiple dimers. However, dimer formation is not biologically relevant; GQs do not interact amongst each other in vivo.

Dimer formation is attributed to the much higher concentration of DNA and packing forces experienced in a crystal structure. This leads to the a diminished area of the GQ being exposed for interactions with K+ present in the solution, not indicative of the state in the cell. The GQ DNA when bound to NMM is monomeric in solution, a representative state of DNA in-vivo. Therefore, the dimer interactions do not attribute to the stability of GQ structure and only arise through experiment conditions required for x-ray crystallography. Fortunately, due to comparisons between other GQ monomeric structures, the dimerization does not significantly change the interactions that are relevant to directly compare this study to the structure in vivo. The formation of the dimer still results in a 1:1 stoichiometry between NMM and GQs in solution, not affecting the information to be gained from analyzing these interactions.5 It is important to focus on the NMM-GQ interactions specifically and not the dimer interactions when studying the ligand effects on stability as a possible telomerase inhibitor anti-cancer drug.

IV. N-methyl mesoporphyrin IX Interactions

One molecule of NMM binds to the 3’ face of the GQ, similar to other anti-cancer ligands that have been analyzed binding to the same GQ DNA sequence. This 3’ face is made available for binding in the parallel GQ conformation, while antiparallel structures have loops that block both the 3’ and 5’ tetrads from ligand binding.5 The macrocycle structure, the inner portion of the ligand, binds to the accessible 3’ tetrad through pi stacking interactions. The portion of NMM is 3.6 angstroms away from the tetrad, which is further than other ligands and indicates that there must be another reason for GQ selectivity.

Another interaction is the N-methyl group of NMM that is bent 44.8 degrees away from the aromatic plane of the ligand towards the K+ ion channel. This causes the ligand core to be off center and asymmetrically bind to the GQ.5 However this could lead to positive interactions between the nitrogens in the ring of NMM and the K+, or it could also benefit side chain interactions with the telomeric DNA grooves. These core interactions and their importance in binding are emphasized by the decreased movement of the core NMM atoms and the increased disorder of the peripheral atoms as indicated by the heat map HeatmapKey .

In addition to these core interactions, the exterior portion of the ligand also interacts in a stereo-specific manner with the GQ structure. The two propinate interact with the . The first interacts through water mediated hydrogen bonding with straddling G3 backbone phosphates.8 The second also interacts via water mediated hydrogen bonding with on the 3' sugar of G22.8

There are no electrostatic attractions between the GQ and NMM. This lessens the strength of binding, with a binding constant of ~105 M-1.5 Although the binding constant is unexceptional, the specificity for parallel GQs is the noteworthy attribute of NMM as an anti-cancer ligand. This specificity arises from the structural adjustment of NMM in order to align with the 3’ tetrad along with the isomerization of the GQ to adopt the parallel conformation. At this point, NMM is a starting point for research towards more selective GQ binding ligands.5 These ligands will lead to anti-cancer drugs with better efficacy towards inhibiting telomerase. Tthe specific GQ binding, will limit side effects from non-specific binding of other molecules or other DNA structures.

V. References

1) Rhodes, Daniela, and Hans J. Lipps. "G-quadruplexes and Their Regulatory Roles in Biology." Nucleic Acids Research 43.18 (2015): 8627-637. Web.

2) Ferreira, Rubén, Adrien Marchand, and Valérie Gabelica. "Mass Spectrometry and Ion Mobility Spectrometry of G-quadruplexes. A Study of Solvent Effects on Dimer Formation and Structural Transitions in the Telomeric DNA Sequence D(TAGGGTTAGGGT)." Methods 57.1 (2012): 56-63.

4) Raffaele, Joseph M. "Telomeres, Telomerase, and TA-65. What You Need to Know in 2013." PhysioAge Medical Group. Lecture. http://www.slideshare.net/telomerescience/telomeres-telomerase-and-ta65>

5) Nicoludis, John M., Stephen T. Miller, Philip D. Jeffrey, Steven P. Barrett, Paul R. Rablen, Thomas J. Lawton, and Liliya A. Yatsunyk. "Optimized End-Stacking Provides Specificity Of N-Methyl Mesoporphyrin IX for Human Telomeric G-Quadruplex DNA." Journal of the American Chemical Society 134.50 (2012): 20446-0456. Web.

6) Beale, S. I., and T. Foley. "Induction of -Aminolevulinic Acid Synthase Activity and Inhibition of Heme Synthesis in Euglena Gracilis by N-Methyl Mesoporphyrin IX." Plant Physiology 69.6 (1982): 1331-333. Web.

7) Bhattacharyya, Debmalya, Gayan Mirihana Arachchilage, and Soumitra Basu. "Metal Cations in G-Quadruplex Folding and Stability." Frontiers in Chemistry 4 (2016). Web.

8) Nicoludis, John M., Stephen T. Miller, Philip D. Jeffrey, Steven P. Barrett, Paul R. Rablen, Thomas J. Lawton, and Liliya A. Yatsunyk. "Supporting Information Optimized End-Stacking Provides Specificity Of N-Methyl Mesoporphyrin IX for Human Telomeric G-Quadruplex DNA."
Journal of the American Chemical Society 134.50 (2012): 20446-0456. Web.

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