Final Report Summary - DNAMI (Aromatic oligoamide foldamers as mimics of double helical DNA)
The general objective of the project entitled ‘Aromatic oligoamide foldamers as mimics of double helical DNA’ is to rationally design and synthesize single helically folded aromatic oligoamides with anionic substituents mimicking the surface of double stranded B-DNA and to demonstrate the ability of these foldamer-based DNA mimics (DNAMIs) to interact with some DNA-binding proteins, primarily Topoisomerase I. This approach can create a new platform to study DNA-protein interactions and to develop novel inhibitors. We anticipate that DNAMIs may pave the way towards new cancer therapeutics. The three following specific objectives of this project were proposed based on the encouraging preliminary results obtained using oligomers having isobutyl (lipophilic) functionalities.
1. Design and synthesis of a mini library of oligomers and identification of the best member (or members) of the oligomer series, which could most accurately mimic the double helical trail of phosphates of DNA.
2. Structural assessment of DNAMIs: implicit in objective 1 is structure determination, in particular using X-ray crystallography, to accurately compare them to the B-DNA structure.
3. Functional assessment of DNAMIs: Identification of proteins that bind to the synthesized DNAMIs and evaluation of the binding of DNAMIs to DNA processing enzymes, primarily Topoisomerase, other enzymes (HIV-IN, helicase, phosphatases, histones) being considered.
Over the two-year duration of the project, progress can be summarized as follows:
a) Monomer Synthesis: two types of quinoline based monomers named Q and mQ having phosphonate functionalities were synthesized. The preparation of 4-hydroxy-quinolines was carried out by addition of substituted anilines on dimethyl acetylene dicarboxylate followed by a thermal cyclization in refluxing Ph2O. Side chain functionality in mQ and Q at position 4 were introduced via Mitsunobu reactions with diethyl (hydroxymethyl) phosphonate. The Boc protected mQ monomer was produced in two steps (one pot) from a nitrile (H2/Ra-Ni then BocOBoc). Main chain acid on Q was protected as trimethylsilylethyl (TMSE) ester, preventing any side reaction with the side chains during the deprotection step. A side chain positional variant with substitution at position 5 on Q was also synthesized. These monomers were used for building up the oligomers by solution phase synthesis.
b) Oligomer synthesis: using monomers at hand, oligomers were synthesized by a ‘segment doubling strategy’. First, a dimeric building block (mQ-Q)n (where, n =1) was built by coupling of Boc-mQ-acid with amino quinoline having main chain acid group protected acid TMSE group. Further, the C-terminal TMSE protection was replaced with benzylic group at the dimeric stage due to the partial loss of orthogonality observed at the deprotection of Boc group on higher oligomers with TFA. Dimeric acid (amino group protected as Boc) was coupled with dimeric amine (main chain acid group protected as benzyl ester) using HBTU, DIPEA yielded the required tetramer. Oligomers of length n = 2, 4, 8, 12 and 16 were synthesized using the same strategy. In order to get the water soluble version of oligomers, diethyl phosphonates were removed by trimethylsilyl bromide, and cation exchange afforded the final oligomers as ammonium phosphonate salts. These were used for the biological experiments.
c) Characterization of oligomers mainly by single crystal X-ray diffraction studies
As we expected, the protected neutral organic oligomers (diethyl phosphonates) were easy to crystallize in a solvent mixture of ethyl acetate and chloroform whereas, the fully water soluble oligomers turned to be bit challenging. The oligomers were crystallized in a solvent mixture of ethyl acetate and dichloromethane. We were able to get crystal structures of (mQQ5)4, mQQ4)8, and (mQQ4)16 (all with side chains protected) and structural analysis validate the design strategy. The oligomers show that substitutents follow a helical trail which very closely resembles the double helical B-DNA structure, with the side chains taking the place of phosphodiester linkages. An overlay of the structure of (mQQ4)16 with a 16-base pair B-DNA shows a very good match in curvature extending to over 8-base pairs, indicating that B-DNA mimicry extends to lengths matching those typically involved in protein-DNA contacts.
d) In vitro functional assays of DNAMIs with Top1.
This part of the project was being carried out in collaboration with the research group of Dr. Philippe Pourquier, medical research unit U916 located at the Comprehensive Cancer Center in Bordeaux (Bergonié Institute). Initial screening results with various DNA binding enzymes show that DNAMIs do not have any effect on activity of enzymes like benzonase, DNAse (these enzymes break polynucleotides into smaller fragments) and also on the nuclease activity. The in vitro testing of the effects of DNAMIs on Top1 catalytic activity was performed using “topoisomerase assays” that have been specifically developed to evaluate the potency of topoisomerase inhibitors. Studies show that oligomer of sufficient length (n > 8) with free phosphonate groups is able to inhibit the binding of Top1 with DNA (IC50 in nano molar range) and the inhibition potency increases with increase in the chain length of oligomers. Results also show that moving the substitution from position 4 to position 5 on Q unit did not change the inhibition efficiency of the oligomer. These experiments were carried out by the researcher after proper training given by Dr. Philippe Pourquier in his lab. The synthesis of radio-labeled DNAMI for gel retardation assay (as proposed in the project) was done by reacting a modified nucleotide (having a spacer with an OSu) to the terminal amino methylene group of the DNAMI.
In summary, the main results of the project ar as follows:
- Accomplished the synthesis of monomers with phosphonate groups for the solution phase synthesis.
- Oligomers (protected with diethyl phosphonates) were synthesized in solution phase by segment doubling strategy and are fully characterized.
-Structural investigation were performed on oligomers (protected with diethyl phosphonates) by single
crystal X-ray diffraction studies.
-The water soluble oligomers were synthesized and purified by HPLC.
-For EMSA studies to have radiolabelled oligomer, nucleotide foldamer conjugate was synthesized.
- In vitro ‘topoisomerase assay’ was performed to evaluate the binding affinity of DNAMIs with Top1 and the results show that DNAMIs of appropriate length (16 mer and above) act as nanomolar inhibitors.
- The inhibition efficiency does not depend on position of substituent but depends on chain length.
Potential impact and use of the results:
Inhibition of Protein-Protein interaction (PPI), Protein-DNA interaction is of therapeutic relevance in particular in cancer therapy. The creation of functionalized synthetic molecule that can perform like DNA with tailored binding affinity to various enzymes will be a breakthrough in chemistry and biology. Although there are literate reports where mimicking the secondary and tertiary structural motifs of proteins utilizing foldamers, the double helical structure of the DNA as a whole has never been mimicked. The development of foldamer based novel technologies is expected to have a very strong impact on health and the design of novel therapeutic and diagnostic agents. A large number of DNA binding proteins are essential to DNA transcription, replication, recombination and/or DNA repair. Topoisomerase, enzyme that catalyzes the changes in the DNA structure by breaking or rejoining the phosphodiester bond, has become popular target for the cancer therapy treatment. Camptothecins (CPT) are the only class of topoisomerase I (top 1) inhibitors approved for the cancer therapy and the search for new anti-cancer agents is still of utmost importance. The biological studies based on topoisomerase assays reveals interesting finding that DNAMIs are nano molar inhibitors on top1 catalytic activity. Thus our approach and the finding resulted from this project can create a new platform to study DNA-protein interactions and to develop novel inhibitors. We anticipate that DNAMIs may pave the way towards new cancer therapeutics.
1. Design and synthesis of a mini library of oligomers and identification of the best member (or members) of the oligomer series, which could most accurately mimic the double helical trail of phosphates of DNA.
2. Structural assessment of DNAMIs: implicit in objective 1 is structure determination, in particular using X-ray crystallography, to accurately compare them to the B-DNA structure.
3. Functional assessment of DNAMIs: Identification of proteins that bind to the synthesized DNAMIs and evaluation of the binding of DNAMIs to DNA processing enzymes, primarily Topoisomerase, other enzymes (HIV-IN, helicase, phosphatases, histones) being considered.
Over the two-year duration of the project, progress can be summarized as follows:
a) Monomer Synthesis: two types of quinoline based monomers named Q and mQ having phosphonate functionalities were synthesized. The preparation of 4-hydroxy-quinolines was carried out by addition of substituted anilines on dimethyl acetylene dicarboxylate followed by a thermal cyclization in refluxing Ph2O. Side chain functionality in mQ and Q at position 4 were introduced via Mitsunobu reactions with diethyl (hydroxymethyl) phosphonate. The Boc protected mQ monomer was produced in two steps (one pot) from a nitrile (H2/Ra-Ni then BocOBoc). Main chain acid on Q was protected as trimethylsilylethyl (TMSE) ester, preventing any side reaction with the side chains during the deprotection step. A side chain positional variant with substitution at position 5 on Q was also synthesized. These monomers were used for building up the oligomers by solution phase synthesis.
b) Oligomer synthesis: using monomers at hand, oligomers were synthesized by a ‘segment doubling strategy’. First, a dimeric building block (mQ-Q)n (where, n =1) was built by coupling of Boc-mQ-acid with amino quinoline having main chain acid group protected acid TMSE group. Further, the C-terminal TMSE protection was replaced with benzylic group at the dimeric stage due to the partial loss of orthogonality observed at the deprotection of Boc group on higher oligomers with TFA. Dimeric acid (amino group protected as Boc) was coupled with dimeric amine (main chain acid group protected as benzyl ester) using HBTU, DIPEA yielded the required tetramer. Oligomers of length n = 2, 4, 8, 12 and 16 were synthesized using the same strategy. In order to get the water soluble version of oligomers, diethyl phosphonates were removed by trimethylsilyl bromide, and cation exchange afforded the final oligomers as ammonium phosphonate salts. These were used for the biological experiments.
c) Characterization of oligomers mainly by single crystal X-ray diffraction studies
As we expected, the protected neutral organic oligomers (diethyl phosphonates) were easy to crystallize in a solvent mixture of ethyl acetate and chloroform whereas, the fully water soluble oligomers turned to be bit challenging. The oligomers were crystallized in a solvent mixture of ethyl acetate and dichloromethane. We were able to get crystal structures of (mQQ5)4, mQQ4)8, and (mQQ4)16 (all with side chains protected) and structural analysis validate the design strategy. The oligomers show that substitutents follow a helical trail which very closely resembles the double helical B-DNA structure, with the side chains taking the place of phosphodiester linkages. An overlay of the structure of (mQQ4)16 with a 16-base pair B-DNA shows a very good match in curvature extending to over 8-base pairs, indicating that B-DNA mimicry extends to lengths matching those typically involved in protein-DNA contacts.
d) In vitro functional assays of DNAMIs with Top1.
This part of the project was being carried out in collaboration with the research group of Dr. Philippe Pourquier, medical research unit U916 located at the Comprehensive Cancer Center in Bordeaux (Bergonié Institute). Initial screening results with various DNA binding enzymes show that DNAMIs do not have any effect on activity of enzymes like benzonase, DNAse (these enzymes break polynucleotides into smaller fragments) and also on the nuclease activity. The in vitro testing of the effects of DNAMIs on Top1 catalytic activity was performed using “topoisomerase assays” that have been specifically developed to evaluate the potency of topoisomerase inhibitors. Studies show that oligomer of sufficient length (n > 8) with free phosphonate groups is able to inhibit the binding of Top1 with DNA (IC50 in nano molar range) and the inhibition potency increases with increase in the chain length of oligomers. Results also show that moving the substitution from position 4 to position 5 on Q unit did not change the inhibition efficiency of the oligomer. These experiments were carried out by the researcher after proper training given by Dr. Philippe Pourquier in his lab. The synthesis of radio-labeled DNAMI for gel retardation assay (as proposed in the project) was done by reacting a modified nucleotide (having a spacer with an OSu) to the terminal amino methylene group of the DNAMI.
In summary, the main results of the project ar as follows:
- Accomplished the synthesis of monomers with phosphonate groups for the solution phase synthesis.
- Oligomers (protected with diethyl phosphonates) were synthesized in solution phase by segment doubling strategy and are fully characterized.
-Structural investigation were performed on oligomers (protected with diethyl phosphonates) by single
crystal X-ray diffraction studies.
-The water soluble oligomers were synthesized and purified by HPLC.
-For EMSA studies to have radiolabelled oligomer, nucleotide foldamer conjugate was synthesized.
- In vitro ‘topoisomerase assay’ was performed to evaluate the binding affinity of DNAMIs with Top1 and the results show that DNAMIs of appropriate length (16 mer and above) act as nanomolar inhibitors.
- The inhibition efficiency does not depend on position of substituent but depends on chain length.
Potential impact and use of the results:
Inhibition of Protein-Protein interaction (PPI), Protein-DNA interaction is of therapeutic relevance in particular in cancer therapy. The creation of functionalized synthetic molecule that can perform like DNA with tailored binding affinity to various enzymes will be a breakthrough in chemistry and biology. Although there are literate reports where mimicking the secondary and tertiary structural motifs of proteins utilizing foldamers, the double helical structure of the DNA as a whole has never been mimicked. The development of foldamer based novel technologies is expected to have a very strong impact on health and the design of novel therapeutic and diagnostic agents. A large number of DNA binding proteins are essential to DNA transcription, replication, recombination and/or DNA repair. Topoisomerase, enzyme that catalyzes the changes in the DNA structure by breaking or rejoining the phosphodiester bond, has become popular target for the cancer therapy treatment. Camptothecins (CPT) are the only class of topoisomerase I (top 1) inhibitors approved for the cancer therapy and the search for new anti-cancer agents is still of utmost importance. The biological studies based on topoisomerase assays reveals interesting finding that DNAMIs are nano molar inhibitors on top1 catalytic activity. Thus our approach and the finding resulted from this project can create a new platform to study DNA-protein interactions and to develop novel inhibitors. We anticipate that DNAMIs may pave the way towards new cancer therapeutics.