Roger Busson

Zebrafish Bioassay-Guided Natural Product Discovery: Isolation of Angiogenesis Inhibitors from East African Medicinal Plants

Natural products represent a significant reservoir of unexplored chemical diversity for early-stage drug discovery. The identification of lead compounds of natural origin would benefit from therapeutically relevant bioassays capable of facilitating the isolation of bioactive molecules from multi-constituent extracts. Towards this end, we developed an in vivo bioassay-guided isolation approach for natural product discovery that combines bioactivity screening in zebrafish embryos with rapid fractionation by analytical thin-layer chromatography (TLC) and initial structural elucidation by high-resolution electrospray mass spectrometry (HRESIMS). Bioactivity screening of East African medicinal plant extracts using fli-1:EGFP transgenic zebrafish embryos identified Oxygonum sinuatum and Plectranthus barbatus as inhibiting vascular development. Zebrafish bioassay-guided fractionation identified the active components of these plants as emodin, an inhibitor of the protein kinase CK2, and coleon A lactone, a rare abietane diterpenoid with no previously described bioactivity. Both emodin and coleon A lactone inhibited mammalian endothelial cell proliferation, migration, and tube formation in vitro, as well as angiogenesis in the chick chorioallantoic membrane (CAM) assay. These results suggest that the combination of zebrafish bioassays with analytical chromatography methods is an effective strategy for the rapid identification of bioactive natural products.

Solution structure of a HNA–RNA hybrid

BACKGROUND Synthetic nucleic acid analogues with a conformationally restricted sugar-phosphate backbone are widely used in antisense strategies for biomedical and biochemical applications. The modified backbone protects the oligonucleotides against degradation within the living cell, which allows them to form stable duplexes with sequences in target mRNAs with the aim of arresting their translation. The biologically most active antisense oligonucleotides also trigger cleavage of the target RNA through activation of endogenous RNase H. Systematic studies of synthetic oligonucleotides have also been conducted to delineate the origin of the chirality of DNA and RNA that are both composed of D-nucleosides. RESULTS Hexitol nucleic acids (HNA) are the first example of oligonucleotides with a six-membered carbohydrate moiety that can bind strongly and selectively to complementary RNA oligomers. We present the first high resolution nuclear magnetic resonance structure of a HNA oligomer bound to a complementary RNA strand. The HNA-RNA complex forms an anti-parallel heteroduplex and adopts a helical conformation that belongs to the A-type family. Possibly, due to the rigidity of the rigid chair conformation of the six-membered ring both the HNA and RNA strand in the duplex are well defined. The observed absence of end-fraying effects also indicate a reduced conformational flexibility of the HNA-RNA duplex compared to canonical dsRNA or an RNA-DNA duplex. CONCLUSIONS The P-P distance across the minor groove, which is close to A-form, and the rigid conformation of the HNA-RNA complex, explain its resistance towards degradation by Rnase H. The A-form character of the HNA-RNA duplex and the reduced flexibility of the HNA strand is possibly responsible for the stereoselectivity of HNA templates in non-enzymatic replication of oligonucleotides, supporting the theory that nucleosides with six-membered rings could have existed at some stage in molecular evolution.

Acyclic oligonucleotides: possibilities and limitations

Abstract Oligonucleotides containing acyclic nucleosides with a 3( S ),5-dihydroxypentyl ( 1a–e ) or 4( R )-methoxy-3( S ),5-dihydroxypentyl ( 2a ) side chain were prepared and their hybridization properties as well as their stability towards degradation with snake venom posphodiesterase were studied. Attachment of an acyclic nucleoside at the 3′-end of an oligonucleotide makes it extremely resistant against enzymatic breakdown. Whereas oligonucleotides consisting completely of acyclic 2′-deoxyadenosine analogues ( 1a or 2a ) can still hybridize with an unmodified oligothymidylate, completely modified oligothymidylates or hetero-oligomers do not hybridize with their unmodified complementary oligonucleotide. This can be explained by the favourable enthalpy change on hybridization for the oligomers with adenine bases because of their higher degree of stacking and the ability to form T- A * .T triplets. In base-pairing with the natural DNA-nucleosides (dA,dC,dG,T), the acyclic nucleoside analogues ( 1a–e ) discriminate less compared to the natural 2′-deoxynucleosides. 9-(3( S ),5-Dihydroxypentyl)hypoxanthine shows the least spreading in melting temperature on hybridization with the four natural 2′-deoxynucleosides. Because of their conformational flexibility, acyclic nucleosides can be considered as universal nucleoside for the design of probes with ambiguous positions.

SYNTHESIS AND PAIRING PROPERTIES OF OLIGONUCLEOTIDES CONTAINING 3-HYDROXY-4-HYDROXYMETHYL-1-CYCLOHEXANYL NUCLEOSIDES

Cyclohexanyl nucleic acid (CNA) represents a novel enantioselective Watson–Crick base-pairing system. Homochiral oligomers of equivalent chirality show antiparallel Watson–Crick pairing, while those of opposite sense do not. D-CNA hybridizes with natural nucleic acids, preferentially with RNA. A conformational change involving chair inversion is expected to occur upon duplex formation (A→B).

Synthesis of protected D-altritol nucleosides as building blocks for oligonucleotide synthesis

Abstract D-Altritol nucleosides with an adenine and uracil base moiety were obtained by nucleophilic opening of the epoxide ring of 1,5:2,3-dianhydro-4,6- O -benzylidene-D-allitol using the sodium salt of the above mentioned bases. The use of a 2-trimethylsilylethyl protecting group for the O 6 -function of the guanine base offers a useful compromise between stability and acceptable alkylation yields of the N 9 -position if the guanine base. The cytosine nucleoside was synthesized starting from the uracil congener. The 3′-hydroxyl function was protected with a benzoyl group.

Promysalin, a Salicylate-Containing Pseudomonas putida Antibiotic, Promotes Surface Colonization and Selectively Targets Other Pseudomonas

Under control of the Gac regulatory system, Pseudomonas putida RW10S1 produces promysalin to promote its own swarming and biofilm formation, and to selectively inhibit many other pseudomonads, including the opportunistic pathogen Pseudomonas aeruginosa. This amphipathic antibiotic is composed of salicylic acid and 2,8-dihydroxymyristamide bridged by a unique 2-pyrroline-5-carboxyl moiety. In addition to enzymes for salicylic acid synthesis and activation, the biosynthetic gene cluster encodes divergent type II fatty acid biosynthesis components, unusual fatty acid-tailoring enzymes (two Rieske-type oxygenases and an amidotransferase), an enzyme resembling a proline-loading module of nonribosomal peptide synthetases, and the first prokaryotic member of the BAHD family of plant acyltransferases. Identification of biosynthetic intermediates enabled to propose a pathway for synthesis of this bacterial colonization factor.

Synthesis and Evaluation of 6‐Aza‐2′‐deoxyuridine Monophosphate Analogs as Inhibitors of Thymidylate Synthases, and as Substrates or Inhibitors of Thymidine Monophosphate Kinase in Mycobacterium tuberculosis

A series of 5‐substituted analogs of 6‐aza‐2′‐deoxyuridine 5′‐monophosphate, 6‐aza‐dUMP, has been synthesized and evaluated as potential inhibitors of the two mycobacterial thymidylate synthases (i.e., a flavin‐dependent thymidylate synthase, ThyX, and a classical thymidylate synthase, ThyA). Replacement of C(6) of the natural substrate dUMP by a N‐atom in 6‐aza‐dUMP 1a led to a derivative with weak ThyX inhibitory activity (33% inhibition at 50 μM). Introduction of alkyl and aryl groups at C(5) of 1a resulted in complete loss of inhibitory activity, whereas the attachment of a 3‐(octanamido)prop‐1‐ynyl side chain in derivative 3 retained the weak level of mycobacterial ThyX inhibition (40% inhibition at 50 μM). None of the synthesized derivatives displayed any significant inhibitory activity against mycobacterial ThyA. The compounds have also been evaluated as potential inhibitors of mycobacterial thymidine monophosphate kinase (TMPKmt). None of the derivatives showed any significant TMPKmt inhibition. However, replacement of C(6) of the natural substrate (dTMP) by a N‐atom furnished 6‐aza‐dTMP (1b), which still was recognized as a substrate by TMPKmt.

Synthesis and Properties of Aminopropyl Nucleic Acids

Oligonucleotides that contain up to three aminopropyl nucleoside analogues have been synthesized. Dimers of aminopropyl adenine and thymidine were prepared and used as building blocks by applying phosphoramidite chemistry. Both R and S isomers of the aminopropyl nucleosides were used. This incorporation led to a reduction of thermal stability of double‐stranded DNA. Furthermore, the (R)‐adenine analogue, which yielded (S)‐APNA, can be considered as a candidate for universal base pairing.

Synthesis of 2′-Deoxy-2′ -Fluoro-D-Arabinopyranopyranosyl Nucleosides and Their 3′,4′-Seco analogues

Abstract 2′-Deoxy-2′-fluoro-D-arabinopyranosyl nucleosides were synthesized by condensation of 1,3,4-tri-O-benzoyl-2-deoxy-2-fluoro-D-arabinopyranose with the appropriate silylated bases in the presence of trimethylsilyl triflate. Scission of the 3′,4′-bond by periodate oxidation followed by sodium borohydride reduction resulted in the formation of the 3′,4′-seco analogues of the 2′-deoxy-2′-fluoro-D-arabinofuranosyl nucleosides.

Homo-N-nucleosides: Incorporation into oligonucleotides and antiviral activity

Abstract Homo-N-nucleosides can be efficiently synthesized from 2-deoxyribose. When incorporated in an oligonucleotide, the compounds have a detrimental influence on duplex stability and on the catalytic activity of hammerhead ribozymes. However, homo-N-nucleosides with a guanine or adenine base moiety do exhibit selective antiviral activity against herpes simplex virus (HSV-1 and HSV-2).