Erik B. Pedersen

Non-nucleoside reverse transcriptase inhibitors: the NNRTI boom

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are promising drugs for the treatment of HIV when used in combination with other anti-HIV drugs such as nucleoside reverse transcriptase (RT) inhibitors and protease inhibitors. The first generation of NNRTIs have, however, suffered from the rapid development of resistance. This review discusses the properties of the FDA-approved NNRTI drugs and focuses on the recent efforts being made to produce second generation inhibitors that circumvent this resistance problem.

Identification of a New G-Quadruplex Motif in the KRAS Promoter and Design of Pyrene-Modified G4-Decoys with Antiproliferative Activity in Pancreatic Cancer Cells

A new quadruplex motif located in the promoter of the human KRAS gene, within a nuclease hypersensitive element (NHE), has been characterized. Oligonucleotides mimicking this quadruplex are found to compete with a DNA-protein complex between NHE and a nuclear extract from pancreatic cancer cells. When modified with (R)-1-O-[4-1-(1-pyrenylethynyl) phenylmethyl]glycerol insertions (TINA), the quadruplex oligonucleotides showed a dramatic increase of the T(m) (deltaT(m) from 22 to 32 degrees C) and a strong antiproliferative effects in Panc-1 cells.

G4-DNA Formation in the HRAS Promoter and Rational Design of Decoy Oligonucleotides for Cancer Therapy

HRAS is a proto-oncogene involved in the tumorigenesis of urinary bladder cancer. In the HRAS promoter we identified two G-rich elements, hras-1 and hras-2, that fold, respectively, into an antiparallel and a parallel quadruplex (qhras-1, qhras-2). When we introduced in sequence hras-1 or hras-2 two point mutations that block quadruplex formation, transcription increased 5-fold, but when we stabilized the G-quadruplexes by guanidinium phthalocyanines, transcription decreased to 20% of control. By ChIP we found that sequence hras-1 is bound only by MAZ, while hras-2 is bound by MAZ and Sp1: two transcription factors recognizing guanine boxes. We also discovered by EMSA that recombinant MAZ-GST binds to both HRAS quadruplexes, while Sp1-GST only binds to qhras-1. The over-expression of MAZ and Sp1 synergistically activates HRAS transcription, while silencing each gene by RNAi results in a strong down-regulation of transcription. All these data indicate that the HRAS G-quadruplexes behave as transcription repressors. Finally, we designed decoy oligonucleotides mimicking the HRAS quadruplexes, bearing (R)-1-O-[4-(1-Pyrenylethynyl) phenylmethyl] glycerol and LNA modifications to increase their stability and nuclease resistance (G4-decoys). The G4-decoys repressed HRAS transcription and caused a strong antiproliferative effect, mediated by apoptosis, in T24 bladder cancer cells where HRAS is mutated.

MAZ-binding G4-decoy with locked nucleic acid and twisted intercalating nucleic acid modifications suppresses KRAS in pancreatic cancer cells and delays tumor growth in mice

KRAS mutations are primary genetic lesions leading to pancreatic cancer. The promoter of human KRAS contains a nuclease-hypersensitive element (NHE) that can fold in G4-DNA structures binding to nuclear proteins, including MAZ (myc-associated zinc-finger). Here, we report that MAZ activates KRAS transcription. To knockdown oncogenic KRAS in pancreatic cancer cells, we designed oligonucleotides that mimic one of the G-quadruplexes formed by NHE (G4-decoys). To increase their nuclease resistance, two locked nucleic acid (LNA) modifications were introduced at the 3′-end, whereas to enhance the folding and stability, two polycyclic aromatic hydrocarbon units (TINA or AMANY) were inserted internally, to cap the quadruplex. The most active G4-decoy (2998), which had two para-TINAs, strongly suppressed KRAS expression in Panc-1 cells. It also repressed their metabolic activity (IC50 = 520 nM), and it inhibited cell growth and colony formation by activating apoptosis. We finally injected 2998 and control oligonucleotides 5153, 5154 (2 nmol/mouse) intratumorally in SCID mice bearing a Panc-1 xenograft. After three treatments, 2998 reduced tumor xenograft growth by 64% compared with control and increased the Kaplan–Meier median survival time by 70%. Together, our data show that MAZ-specific G4-decoys mimicking a KRAS quadruplex are promising for pancreatic cancer therapy.

1‐, 2‐, and 4‐Ethynylpyrenes in the Structure of Twisted Intercalating Nucleic Acids: Structure, Thermal Stability, and Fluorescence Relationship

A postsynthetic, on-column Sonogashira reaction was applied on DNA molecules modified by 2- or 4-iodophenylmethylglycerol in the middle of the sequence, to give the corresponding ortho- and para-twisted intercalating nucleic acids (TINA) with 1-, 2-, and 4-ethynylpyrene residues. The convenient synthesis of 2- and 4-ethynylpyrenes started from the hydrogenolysis of pyrene that has had the sulfur removed and separation of 4,5,9,10-tetrahydropyrene and 1,2,3,6,7,8-hexahydropyrene, which were later converted to the final compounds by successive Friedel-Crafts acetylation, aromatization by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, and a Vilsmeier-Haack-Arnold transformation followed by a Bodendorf fragmentation. Significant alterations in thermal stability of parallel triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in homopyrimidine TINAs. Thus, for para-TINAs the bulge insertion of an intercalator led to high thermal stability of Hoogsteen-type parallel triplexes and duplexes, whereas Watson-Crick-type duplexes were destabilized. In the case of ortho-TINA, both Hoogsteen and Watson-Crick-type complexes were stabilized. Alterations in the thermal stability were highly influenced by the ethynylpyrene isomers used. This also led to TINAs with different changes in fluorescence spectra depending on the secondary structures formed. Stokes shift of approximately 100 nm was detected for pyren-2-ylethynylphenyl derivatives, whereas values for 1- and 4-ethynylpyrenylphenyl conjugates were 10 and 40 nm, respectively. In contrast with para-TINAs, insertion of two ortho-TINAs opposite each other in the duplex as a pseudo-pair resulted in formation of an excimer band at 505 nm for both 1- and 4-ethynylpyrene analogues, which was also accompanied with higher thermal stability.

Synthesis of an aza analogue of 2-deoxy-D-ribofuranose and its homologues.

Azasugars were obtained in one-pot reactions by catalytic reduction reactions of amino group precursors in aldosugars followed by intramolecular reductive amino alkylation reactions. (3R,4S)-4-[(1S)-1,2-Dihydroxyethyl]pyrrolidin-3-ol was obtained from D-xylose by two different strategies through 3-C-cyano-3-deoxy-D-ribo-pentofuranose or 3-C-azidomethyl-3-deoxy-D-ribo-pentofuranose in 6 and 16% overall yields, respectively. The oxidative cleavage of the diol group in the corresponding Fmoc-azasugar followed by deprotection afforded (3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol. (3R,4S)-4-[(1S,2R)-1,2,3-Trihydroxypropyl]pyrrolidin-3-ol was synthesized from diacetone-D-glucose through 3-deoxy-3-C-nitromethyl-D-allose and the overall yield was 7%.

An unusual hexafluorosilicato-bridged chain compound; crystal structure of catena-(μ-Hexafluorosilicato)-tetrakis(5-phenylpyrazole)copper(II)

Abstract The reaction of a methanolic solution of M(II)(BF 4 ) 2 salts and 3(5)-phenylpyrazool (ppzH) in glass vessels yields chain compounds of general formula M(II)(ppzH) 4 (SiF 6 ) (M  Co, Ni, Cu), in which the SiF 6 2− group is the bridging ligand. The compounds can also be prepared starting from the hydrated hexafluorosilicates. The structure of one of these compounds, i.e. catena -(μ-hexafluorosilicato)-tetrakis(5-phenylpyrazole)copper(II) Cu(C 9 H 8 N 2 ) 4 (SiF 6 ) ( I ) has been determined from X-ray diffraction data. The crystals are triclinic, space group P 1 lattice constants a =5.531(7), b =12.211(5), c =12.140(7) A, α=121.69(5), β=99.30(4), γ=101.73(3)°, Z =2. The Cu(II) ions are at special positions 0,0,0 and 1 2 , 1 2 , 1 2 and are linked by bridging SiF 6 2− groups. The copper ions are coordinated by four pyrazole nitrogen atoms and the fluorine atoms of the SiF 6 2− group as axial ligands. CuN pyr distances are 2.002(5) and 1.992(5) A for Cu(1) and 2.009(5) and 1.994(5) A for Cu(2). CuF distances are 2.251(3) A for Cu(1) and 2.245(3) A for Cu(2). Magnetic susceptibility measurements of I and the analog compounds Ni(C 9 H 8 N 2 ) 4 (SiF 6 ) ( II ) and Co(C 9 H 8 N 2 ) 4 (SiF 6 ) ( III ) show no significant magnetic interaction. The magnetic and spectroscopic properties of the compounds are discussed in relation to their structure.

Synthesis of 1′-aza-C-nucleosides from (3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol

Abstract Pyrimidine 1′-aza-C-nucleosides are synthesised by the fusion of 5-bromouracil, 5-bromocytosine and 5-bromoisocytosine with (3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol in 40–41% yield. A homologue of 1′-aza-Ψ-uridine is obtained in a Mannich reaction in 65% yield by treatment of the azasugar, paraformaldehyde and uracil. N-Alkylation of (3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol with 6-chloromethyluracil gives the 6-regioisomeric homologue. (3R,4R)-4-(Hydroxymethyl)pyrrolidin-3-ol is synthesised in 25% overall yield from diacetone- d -glucose via 3-C-(azidomethyl)-3-deoxy- d -allose which is subjected to an intramolecular reductive amino alkylation reaction to give (3R,4S)-4-[(1S,2R)-1,2,3-trihydroxypropyl]pyrrolidin-3-ol followed by Fmoc protection, oxidative cleavage of the triol group with further reduction of the obtained aldehyde and subsequent deprotection of the nitrogen atom.

Intercalating nucleic acids (INAs) with insertion of N-(pyren-1-ylmethyl)-(3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol. DNA (RNA) duplex and DNA three-way junction stabilities

N-(Pyren-1-ylmethyl)-(3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol was synthesised from (3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol and (3R,4S)-4-[(1S)-1,2-dihydroxyethyl] pyrrolidin-3-ol using alkylation with 1-(chloromethyl)pyrene or reductive amination with pyrene-1-carbaldehyde and NaCNBH3. The incorporation of N-(pyren-1-ylmethyl)azasugar moiety into oligodeoxynucleotides (ODN) as a bulge to form an intercalating nucleic acid (INA) induced a slight destabilization of INA-DNA duplex, whereas the INA-RNA duplex was strongly destabilized and 9 degrees C difference per modification in thermal stability between INA-DNA over INA-RNA duplexes was observed. The stabilization of a DNA three way junction (TWJ) was improved when the intercalator moiety was inserted into the junction region as a bulge.

Triplex Formation by Pyrene-Labelled Probes for Nucleic Acid Detection in Fluorescence Assays

Triplex‐forming homopyrimidine oligonucleotides containing insertions of a 2′–5′ uridine linkage featuring a pyrene moiety at the 3′‐position exhibit strong fluorescence enhancement upon binding to double‐stranded DNA through Hoogsteen base pairing. It is shown that perfect matching of the new modification to the base pair in the duplex is a prerequisite for strong fluorescence, thus offering the potential to detect single mutations in purine stretches of duplex DNA. The increase in the fluorescence signal was dependent on the thermal stability of the parallel triplex, so a reduction in the pH from 6.0 to 5.0 resulted in an increase in thermal stability from 25.0 to 55.0 °C and in an increase in the fluorescence quantum yield (ΦF) from 0.061 to 0.179, while the probe alone was fluorescently silent (ΦF=0.001–0.004). To achieve higher triplex stability, five nucleobases in a 14‐mer sequence were substituted with α‐L‐LNA monomers, which provided a triplex with a Tm of 49.5 °C and a ΦF of 0.158 at pH 6.0. Under similar conditions, a Watson–Crick‐type duplex formed with the latter probe showed lower fluorescence intensity (ΦF=0.081) than for the triplex.