Frédéric Godde

Solid phase synthesis of aromatic oligoamides: application to helical water-soluble foldamers.

Synthetic helical aromatic amide foldamers and in particular those based on quinolines have recently attracted much interest due to their capacity to adopt bioinspired folded conformations that are highly stable and predictable. Additionally, the introduction of water-solubilizing side chains has allowed to evidence promising biological activities. It has also created the need for methods that may allow the parallel synthesis and screening of oligomers. Here, we describe the application of solid phase synthesis to speed up oligomer preparation and allow the introduction of various side chains. The synthesis of quinoline-based monomers bearing protected side chains is described along with conditions for activation, coupling, and deprotection on solid phase, followed by resin cleavage, side-chain deprotection, and HPLC purification. Oligomers having up to 8 units were thus synthesized. We found that solid phase synthesis is notably improved upon reducing resin loading and by applying microwave irradiation. We also demonstrate that the introduction of monomers bearing benzylic amines such as 6-aminomethyl-2-pyridinecarboxylic acid within the sequences of oligoquinolines make it possible to achieve couplings using a standard peptide coupling agent and constitute an interesting alternative to the use of acid chloride activation required by quinoline residues. The synthesis of a tetradecameric sequence was thus smoothly carried out. NMR solution structural studies show that these alternate aminomethyl-pyridine residues do not perturb the canonical helix folding of quinoline monomers in protic solvents, contrary to what was previously observed in nonprotic solvents.

Synthesis and evaluation of the antiproliferative activity of novel pyrrolo[1,2-a]quinoxaline derivatives, potential inhibitors of Akt kinase. Part II.

Attenuation of protein kinases by selective inhibitors is an extremely active field of activity in anticancer drug development. Therefore, Akt, a serine/threonine protein kinase, also known as protein kinase B (PKB), represents an attractive potential target for therapeutic intervention. Recent efforts in the development and biological evaluation of small molecule inhibitors of Akt have led to the identification of novel inhibitors with various heterocycle scaffolds. Based on previous results obtained on the antiproliferative activities of new pyrrolo[1,2-a]quinoxalines, a novel series was designed and synthesized from various substituted phenyl-1H-pyrrole-2-carboxylic acid alkyl esters via a multistep heterocyclization process. These new compounds were tested for their in vitro ability to inhibit the proliferation of the human leukemic cell lines K562, U937, and HL60, and the breast cancer cell line MCF7. The first biological evaluation of our new substituted pyrrolo[1,2-a]quinoxalines showed antiproliferative activity against the tested cell lines. From a general SAR point of view, these preliminary biological results highlight the importance of substitution at the C-4 position of the pyrroloquinoxaline scaffold by a benzylpiperidinyl fluorobenzimidazole group, and also the need for a functionalization on the pyrrole ring.

G-Quadruplex DNA Bound by a Synthetic Ligand is Highly Dynamic

Using single molecule fluorescence resonance energy transfer, we investigated the interaction between a quadruplex-binding ligand and the human telomeric G-quadruplex. The binding of quinolinecarboxamide macrocycle to telomeric DNA was essentially irreversible and selectively induced and favored one quadruplex conformation. The ligand−quadruplex complex displayed intramolecular dynamics including quadruplex folding and unfolding in the absence of ligand association and dissociation. We report that the G-quadruplex can be stabilized without preventing the intrinsic intramolecular dynamics of telomeric DNA.

Use of the Fluorescent Nucleoside Analogue Benzo[g]quinazoline 2′-O-Methyl--D-ribofuranoside to Monitor the Binding of the HIV-1 Tat Protein or of Antisense Oligonucleotides to the TAR RNA Stem-Loop

The Tat protein is an essential trans-activator of HIV gene expression. It interacts with its RNA recognition sequence, the trans-activation responsive region TAR, as well as cellular factors. These interactions are potential targets for drug discovery against HIV infection. We have developed a new and sensitive assay for the measurement of Tat binding to TAR in solution under equilibrium conditions based on the change of fluorescence of the base analogue benzo[g]quinazoline-2,4(1H,3H)-dione (BgQ) incorporated into the chemically synthesized model TAR stem-loop 2 to which was added Tat-[37-72] peptide (3). The results show that Tat-TAR binding strength is 2 – 3-fold stronger than has previously been determined by mobility-shift analysis. Changes of fluorescence were used also to measure the binding of antisense 2′-O-methyloligonucleotides to TAR 2.

Targeting DNA G-Quadruplexes with Helical Small Molecules

We previously identified quinoline‐based oligoamide helical foldamers and a trimeric macrocycle as selective ligands of DNA quadruplexes. Their helical structures might permit targeting of the backbone loops and grooves of G‐quadruplexes instead of the G‐tetrads. Given the vast array of morphologies G‐quadruplex structures can adopt, this might be a way to achieve sequence selective binding. Here, we describe the design and synthesis of molecules based on macrocyclic and helically folded oligoamides. We tested their ability to interact with the human telomeric G‐quadruplex and an array of promoter G‐quadruplexes by using FRET melting assay and single‐molecule FRET. Our results show that they constitute very potent ligands—comparable to the best so far reported. Their modes of interaction differ from those of traditional tetrad binders, thus opening avenues for the development of molecules specific for certain G‐quadruplex conformations.

How can folded biopolymers and synthetic foldamers recognize each other

In the last decade, chemists have synthesized numerous families of oligomeric foldamers by using a variety of backbones. In terms of their chemical composition, foldamers may be quite remote from biopolymers, yet their folded motifs—helices, turns, linear strands— often resemble those of their natural counterparts. Such a resemblance opens great perspectives for the future development of foldamers. Indeed, the control of molecular structure and dynamics through folding is the corner stone of biopolymers’ extraordinary functions. The use of artificial backbones to invent new functions is thus expected to give access to countless applications. Some long-term objectives, such as artificial enzymes from non-a-peptidic backbones or molecular systems that would store and copy information as efficiently as DNA, are being considered but remain far from being achieved, even though the first artificial tertiary and quaternary structures and even catalytic activities have recently been reported. Nonetheless, other foldamer properties have been intensely investigated in recent years, in particular their potential to interfere with biological functions. At first glance, the idea of using synthetic foldamers as potential therapeutic agents is not obvious, because they are generally much larger—typically between 0.5 and 5 kDa—than conventional drugs. However, size can also help solve problems that are hard to tackle with smaller molecules. Besides, the rapid development of biologics shows that entire proteins and even cells and tissues may be used as medicinal products. Moreover, foldamers tend to show high resistance to enzyme degradation and may advantageously replace a-peptides or oligonucleotides as active substances. So why wouldn’t foldamers find their own niche? One area where a large size and the plasticity of a folded structure become an advantage is in binding to protein or nucleic acid surfaces and the subsequent inhibition of interactions between biopolymers. The sites at which these interactions take place have long been recognized as valid targets, but ACHTUNGTRENNUNGefficient binding at biopolymer surfaces with small molecules has met limited success so far. On the other hand, foldamers have been shown to fold into tertiary and quaternary structures in which artificial secondary motifs bind to other artificial secondary motifs. Foldamers thus also emerge as suitable candidates for binding to the surfaces of protein or nucleic acid folded conformations. A growing number of publications validate this concept. Examples that date from even before the term “foldamer” was coined include numerous mimics of oligonucleotidic backbones, such as peptide nucleic acids (PNA), which were designed in the context of the socalled antisense and antigene strategies ; peptoids, which are a-peptidic oligomers whose side chains have been shifted to the amide nitrogen atoms; and oligoACHTUNGTRENNUNGamides of pyrrole and imidazole, which bind to the minor groove of duplex DNA. In the past few years, aromatic oligoamide foldamers with helical or linear conformations, 11] artificial a-peptidic constructs, such as mini-proteins, branched structures, or macrocycles, 15] b-peptide helices, and helical a/b-peptide hybrids have all been shown to bind to protein or nucleic acid structures and are presumed to cover a substantial part of the surface areas of their targets. Two approaches may be envisaged to identifying synthetic folded oligomers that recognize biopolymer targets. The first consists of eliciting arrays of interactions that do not exist in nature, and creating de novo new recognition motifs. Although there is no fundamental obstacle to this approach, examples of the sort are quite rare. Quinoline-derived helical oligoamides have been serendipitously found to bind to G-quadruplexes, and their mode of interaction is likely to be new but it has not yet been elucidated. Pyrrole and imidazole oligoACHTUNGTRENNUNGamides that bind in the minor groove of DNA were initially inspired by the binding mode of the natural product distamycin A, but were thereupon extended by design to an artificial extensive recognition mode of nucleotidic sequences. In fact, most artificial folded structures capable of recognizing biopolymers through new recognition schemes are not synthetic foldamers but non-natural peptidic or nucleotidic sequences, such as aptamers, that are produced by in vitro evolution techniques using molecular biology tools. The second strategy for identifying synthetic folded oligomers that recognize biopolymer targets consists of mimicking arrays of interactions that already exist in nature, for example protein epitopes. This generally amounts to creating mimics of the secondary-folded motifs of proteins or nucleic acids. Some of these mimics follow quite obvious ACHTUNGTRENNUNGdesigns: PNAs as DNA analogues and peptoids as peptide analogues. Yet the task is far from easy. For example, a central issue that has proven to be very challenging is the design of a-helical peptide mimics. This problem is often re[a] Dr. F. Godde, Dr. I. Huc Institut Europ en de Chimie et Biologie Universit de Bordeaux–CNRS UMR 5248 2 rue Robert Escarpit, 33607 Pessac (France) Fax: (+ 33) 540-002-215 E-mail : i.huc@iecb.u-bordeaux.fr [b] B. Baptiste Laboratoire de Pharmacochimie Universit de Bordeaux 146 rue L o Saignat, 33076 Bordeaux (France)

Single helically folded aromatic oligoamides that mimic the charge surface of double-stranded B-DNA

Numerous essential biomolecular processes require the recognition of DNA surface features by proteins. Molecules mimicking these features could potentially act as decoys and interfere with pharmacologically or therapeutically relevant protein–DNA interactions. Although naturally occurring DNA-mimicking proteins have been described, synthetic tunable molecules that mimic the charge surface of double-stranded DNA are not known. Here, we report the design, synthesis and structural characterization of aromatic oligoamides that fold into single helical conformations and display a double helical array of negatively charged residues in positions that match the phosphate moieties in B-DNA. These molecules were able to inhibit several enzymes possessing non-sequence-selective DNA-binding properties, including topoisomerase 1 and HIV-1 integrase, presumably through specific foldamer–protein interactions, whereas sequence-selective enzymes were not inhibited. Such modular and synthetically accessible DNA mimics provide a versatile platform to design novel inhibitors of protein–DNA interactions.Molecules that mimic the charge surface of B-DNA could enable the inhibition of DNA processive enzymes. Now, helically folded aromatic oligoamide scaffolds have been synthesized that display anions at positions similar to that of B-DNA phosphates. These foldamer mimics can recognize some DNA binding proteins and inhibit enzymes such as HIV integrase and topoisomerase 1.

Abstract 4794: Oligoamide-based mimics of double-stranded B-DNA as a new class of DNA topoisomerase I catalytic inhibitors

Since the discovery of Topoisomerase I as a specific target for the treatment of cancers more than 30 years ago, only two inhibitors derived from the natural compound camptothecin (CPT) have been approved in the clinic for the treatment of colon, lung and ovarian cancers: topotecan and irinotecan. The cytotoxicity of these Top1 poisons relies on their capability to stabilize covalent Top1-DNA complexes, leading to replication-mediated DNA double-strand breaks. However tumor cells develop multiple resistance mechanisms that are limiting their efficacy. Though new CPT derivatives and other Top1 poisons with various chemical structures have been developed, they share the same resistance mechanisms. Aside from conventional approaches focus on compounds with higher potency to stabilize Top1-DNA complexes, we investigated the possibility to inhibit Top1 binding to DNA and/or DNA cleavage by using DNA mimics that would act as decoys, a strategy that was never explored before. To this aim, single chain oligoamides composed by iteration of mQQ units (Q: 8-amino-2-quinoline carboxylic acid; mQ: 8-aminomethyl-2-quinoline carboxylic acid) were synthesized. These molecules fold into helices that are stabilized by electrostatic repulsions and hydrogen bonds between the amide functions and endocyclic nitrogen atoms at adjacent residues. When Q and mQ precursors are functionalized by negatively charged residues, the positions of these residues in the folded structure match the position of phosphate residues in duplex B-DNA. Because distances between residues in mQQ and QmQ units are slightly different, it is possible to define a major groove and a minor groove, as in B-DNA, with the possibility to impact on groove width by changing the positions of the substituents (Q4 or Q5 for substitution in position 4 or 5 of the quinoline monomer, respectively), clearly establishing (mQQ5)n and (mQQ4)n as potential DNA-mimics of an unprecedented kind. We found that both (mQQ5)n and (mQQ4)n oligomers inhibited the activity of Top1-mediated relaxation of supercoiled DNA in vitro. Inhibition increased with the length of the oligoamide to reach an IC50 value (concentration inhibiting 50% of Top1-mediated DNA relaxation) around the nM range for the longest oligomer that was tested i.e. (mQQ4)16, corresponding to a double-strand DNA of 16 base pairs. By comparison, CPT had an IC50 of ∼10 μM in the same conditions. Top1 inhibition was rather selective as a (mQQ4)8 oligomer moderately inhibited Top2-mediated activity and did not affect the activity of various DNA interacting enzymes such as restriction enzymes XhoI and NdeI or nucleases such as DNAse I, S1 nuclease, benzonase or Flap-endonuclease I. Because these oligomers are resistant to proteases and nucleases and can be synthesized rapidly by solid phase synthesis with the possibility to modify its substituents without altering their helicity, they represent good candidates for drug development. Citation Format: Philippe Pourquier, Krzysztof Ziach, Celine Chollet, Vincent Parissi, Mathieu Marchivie, Panchami Prabhakaran, Partha P. Bose, Katta Laxmi-Reddy, Frederic Godde, Stephane Chaignepain, Jean Marie Schmitter, Ivan Huc. Oligoamide-based mimics of double-stranded B-DNA as a new class of DNA topoisomerase I catalytic inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4794.