Highly stable hexitol based XNA aptamers targeting the vascular endothelial growth factor.

Biomedical applications of nucleic acid aptamers are limited by their rapid degradation in biological fluids and generally demand tedious post-selection modifications that might compromise binding. One possible solution to warrant biostability is to directly evolve chemically modified aptamers from xenobiotic nucleic acids (XNAs). We have isolated fully modified 2'-O-methyl-ribose-1,5-anhydrohexitol nucleic acid (MeORNA-HNA) aptamers targeting the rat vascular endothelial growth factor 164 (rVEGF164). Three sequences have been identified that interact with the target protein with affinities in the low-nanomolar range and HNA modifications appeared to be mandatory for their tight binding. The evolution of these XNA aptamers was accomplished using an in vitro selection procedure starting from a fully sugar-modified library containing a 20mer 2'-OMe-ribonucleotide region followed by a 47mer HNA sequence. The high binding affinity and selectivity of the selected aptamers were confirmed by several methods including gel-shift, fluorescence polarisation, and enzyme-linked oligonucleotide assays. The isolated HNA ligands exhibited higher specificity to the rVEGF164 and human VEGF165 isoforms compared to rat VEGF120, while very low binding efficiencies were observed to streptavidin and thrombin. Furthermore, it was clearly demonstrated that the resulting aptamers possessed a superior stability to degradation in human serum and DNase I solutions.

Synthesis and Structure-Activity Relationship Studies of Benzo[b][1,4]oxazin-3(4H)-one Analogues as Inhibitors of Mycobacterial Thymidylate Synthase†X.

Since the discovery of a flavin-dependent thymidylate synthase (ThyX or FDTS) that is absent in humans but crucial for DNA biosynthesis in a diverse group of pathogens, the enzyme has been pursued for the development of new antibacterial agents against Mycobacterium tuberculosis, the causative agent of the widespread infectious disease tuberculosis (TB). In response to a growing need for more effective anti-TB drugs, we have built upon our previous screening efforts and report herein an optimization campaign of a novel series of inhibitors with a unique inhibition profile. The inhibitors display competitive inhibition toward the methylene tetrahydrofolate cofactor of ThyX, enabling us to generate a model of the compounds bound to their target, thus offering insight into their structure-activity relationships.

Kinetic analysis of N-alkylaryl carboxamide hexitol nucleotides as substrates for evolved polymerases.

Six 1',5'-anhydrohexitol uridine triphosphates were synthesized with aromatic substitutions appended via a carboxamide linker to the 5-position of their bases. An improved method for obtaining such 5-substituted hexitol nucleosides and nucleotides is described. The incorporation profile of the nucleotide analogues into a DNA duplex overhang using recently evolved XNA polymerases is compared. Long, mixed HNA sequences featuring the base modifications are generated. The apparent binding affinity of four of the nucleotides to the enzyme, the rate of the chemical step and of product release, plus the specificity constant for the incorporation of these modified nucleotides into a DNA duplex overhang using the HNA polymerase T6G12_I521L are determined via pre-steady-state kinetics. HNA polymers displaying aromatic functional groups could have significant impact on the isolation of stable and high-affinity binders and catalysts, or on the design of nanomaterials.

Enzymatic Incorporation of Modified Purine Nucleotides in DNA.

A series of nucleotide analogues, with a hypoxanthine base moiety (8-aminohypoxanthine, 1-methyl-8-aminohypoxanthine, and 8-oxohypoxanthine), together with 5-methylisocytosine were tested as potential pairing partners of N8 -glycosylated nucleotides with an 8-azaguanine or 8-aza-9-deazaguanine base moiety by using DNA polymerases (incorporation studies). The best results were obtained with the 5-methylisocytosine nucleotide followed by the 1-methyl-8-aminohypoxanthine nucleotide. The experiments demonstrated that small differences in the structure (8-azaguanine versus 8-aza-9-deazaguanine) might lead to significant differences in recognition efficiency and selectivity, base pairing by Hoogsteen recognition at the polymerase level is possible, 8-aza-9-deazaguanine represents a self-complementary base pair, and a correlation exists between in vitro incorporation studies and in vivo recognition by natural bases in Escherichia coli, but this recognition is not absolute (exceptions were observed).

Base-Modified Nucleic Acids as a Powerful Tool for Synthetic Biology and Biotechnology.

The ability of various nucleoside triphosphate analogues of deoxyguanosine and deoxycytidine with 7-deazadeoxyadenosine (A1 ) and 5-chlorodeoxyuridine (T1 ) to serve as substrates for Taq DNA polymerase was evaluated. The triphosphate set composed of A1 , T1 , and 7-deazadeoxyguanosine with either 5-methyldeoxycytidine or 5-fluorodeoxycytidine was successfully employed in the polymerase chain reaction (PCR) of 1.5 kb fragments as well as random oligonucleotide libraries. Another effective combination of triphosphates for the synthesis of a 1 kb PCR product was A1 , T1 , deoxyinosine, and 5-bromodeoxycytidine. In vivo experiments using an antibiotic-resistant gene containing the latter set demonstrated that the bacterial machinery accepts fully modified sequences as genetic templates. Moreover, the ability of the base-modified segments to selectively protect DNA from cleavage by restriction endonucleases was shown. This approach can be used to regulate the endonuclease cleavage pattern.

Discovery of a new Mycobacterium tuberculosis thymidylate synthase X inhibitor with a unique inhibition profile.

Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis (Mtb), is an infection that is responsible for roughly 1.5 million deaths per year. The situation is further complicated by the wide-spread resistance to the existing first- and second-line drugs. As a result of this, it is urgent to develop new drugs to combat the resistant bacteria as well as have lower side effects, which can promote adherence to the treatment regimens. Targeting the de novo synthesis of thymidylate (dTMP) is an important pathway to develop drugs for TB. Although Mtb carries genes for two families of thymidylate synthases (TS), ThyA and ThyX, only ThyX is essential for its normal growth. Both enzymes catalyze the conversion of uridylate (dUMP) to dTMP but employ a different catalytic approach and have different structures. Also, ThyA is the only TS found in humans. This is the rationale for identifying selective inhibitors against ThyX. We exploited the NADPH oxidation to NADP+ step, catalyzed by ThyX, to develop a spectrophotometric biochemical assay. Success of the assay was demonstrated by its effectiveness (average Z'=0.77) and identification of selective ThyX inhibitors. The most potent compound is a tight-binding inhibitor with an IC50 of 710nM. Its mechanism of inhibition is analyzed in relation to the latest findings of ThyX mechanism and substrate and cofactor binding order.

Chemical Morphing of DNA Containing Four Noncanonical Bases.

The ability of alternative nucleic acids, in which all four nucleobases are substituted, to replicate in vitro and to serve as genetic templates in vivo was evaluated. A nucleotide triphosphate set of 5-chloro-2'-deoxyuridine, 7-deaza-2'-deoxyadenosine, 5-fluoro-2'-deoxycytidine, and 7-deaza-2'deoxyguanosine successfully underwent polymerase chain reaction (PCR) amplification using templates of different lengths (57 or 525mer) and Taq or Vent (exo-) DNA polymerases as catalysts. Furthermore, a fully morphed gene encoding a dihydrofolate reductase was generated by PCR using these fully substituted nucleotides and was shown to transform and confer trimethoprim resistance to E. coli. These results demonstrated that fully modified templates were accurately read by the bacterial replication machinery and provide the first example of a long fully modified DNA molecule being functional in vivo.

Syntheses of 5'-Nucleoside Monophosphate Derivatives with Unique Aminal, Hemiaminal, and Hemithioaminal Functionalities: A New Class of 5'-Peptidyl Nucleotides.

A number of synthetically useful transformations have been developed to generate novel 5'-peptidyl nucleoside monophosphate analogues that incorporate sensitive phosphoaminal, -hemiaminal or -hemithioaminal functionalities. The strategies adopted entailed the coupling between dipeptides, which enclose a reactive Cα-functionalized glycine residue and phosphate or phosphorothioate moieties. These developments led to potentially powerful and general methodologies for the preparation of α-phosphorylated pseudopeptides as well as nucleoside monophosphate mimics. The resulting conjugates are of interest for a variety of important applications, which range from drug development to synthetic biology, as pronucleotides or artificial building blocks for the enzymatic synthesis of xenobiotic information systems. The potential of all dipeptide-TMP conjugates as pyrophosphate mimics in the DNA polymerization reaction was tested, and the influence of the nature of the linker was evaluated by in vitro chain elongation assay in the presence of wild-type microbial DNA polymerases.

Synthesis of an apionucleoside family and discovery of a prodrug with anti-HIV activity.

We report the synthesis of a family of D- and L-furano-D-apionucleosides, their 3'-deoxy, as well as their 2',3'-dideoxy analogues with thymine and adenine nucleobases. Single carbon homologation of 1,2-O-isopropylidene-D-glycero-tetrafuranos-3-ulose (15) and optimized glycosylation conditions involving microwave irradiation were key to the successful synthesis of the target compounds. While all target nucleosides failed to show significant antiviral activity, we demonstrated that the triphosphate of 2',3'-deoxy-D-apio-D-furanoadenosine (1), in contrast to that of its D-apio-L-furanose epimer 2, was readily incorporated into a DNA template by HIV reverse transcriptase to act as a DNA chain terminator. This led us to convert adenine derivative 1 into two phosphoramidate prodrugs. ProTide 9b was found active against HIV-1 and HIV-2 (EC50 = 0.5-1.5 µM), indicating that the lack of activity of the parent nucleoside, and possibly also other members of the D-apio-D-furanose nucleoside family must be sought in the inefficient cellular conversion to the monophosphate.

Discovery of an acyclic nucleoside phosphonate that inhibits Mycobacterium tuberculosis ThyX based on the binding mode of a 5-alkynyl substrate analogue.

The urgent need for new antibiotics poses a challenge to target un(der)exploited vital cellular processes. Thymidylate biosynthesis is one such process due to its crucial role in DNA replication and repair. Thymidylate synthases (TS) catalyze a crucial step in the biosynthesis of thymidine 5-triphosphate (TTP), an elementary building block required for DNA synthesis and repair. To date, TS inhibitors have only been successfully applied in anticancer therapy due to their lack of specificity for antimicrobial versus human enzymes. However, the discovery of a new family of TS enzymes (ThyX) in a range of pathogenic bacteria that is structurally and biochemically different from the "classic" TS (ThyA) has opened the possibility to develop selective ThyX inhibitors as potent antimicrobial drugs. Here, the interaction of the known inhibitor 5-(3-octanamidoprop-1yn-1yl)-2'-deoxyuridine-5'-monophosphate (1) with Mycobacterium tuberculosis ThyX enzyme is explored using molecular modeling starting from published crystal structures, with further confirmation through NMR experiments. While the deoxyuridylate (dUMP) moiety of compound 1 occupies the cavity of the natural substrate in ThyX, the rest of the ligand (the "5-alkynyl tail") extends to the outside of the enzyme between two of its four subunits. The hydrophobic pocket that accommodates the alkyl part of the tail is formed by displacement of Tyr 44.C, Tyr 108.A and Lys 165.A. Changes to the resonance of the Lys 165 NH3 group upon ligand binding were monitored in a titration experiment by 2D HISQC NMR. Guided by the results of the modeling and NMR studies, and inspired by the success of acyclic antiviral nucleosides, compounds where a 5-alkynyl uracyl moiety is coupled to an acyclic nucleoside phosphonate (ANP) were synthesized and evaluated. Of the compounds evaluated, sodium (6-(5-(3-octanamidoprop-1-yn-1-yl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)hexyl)phosphonate (3 e) exhibited 43 % of inhibitory effect on ThyX at 50 µM. While only modest activity was achieved, this is the first example of an ANP inhibiting ThyX, and these results can be used to further guide structural modifications to this class to develop more potent compounds with potential application as antibacterial agents acting through a novel mechanism of action.

Glypican-3-mediated inhibition of CD26 by TFPI: a novel mechanism in hematopoietic stem cell homing and maintenance.

Directional migration determines hematopoietic stem/progenitor cell (HSPC) homing, which depends upon the interaction between the chemokine CXCL12 and its receptor CXCR4. CD26 is a widely expressed membrane-bound ectopeptidase that cleaves CXCL12 thereby depleting its chemokine activity. We identified tissue-factor pathway inhibitor (TFPI) as a biological inhibitor of CD26 in murine and human HSPCs. We observed low-level TFPI expression in endothelial cells in the bone marrow (BM), which did not increase following radiation injury. Treatment of HSPCs with TFPI in vitro led to enhanced HSPC migration toward CXCL12, as well as homing and engraftment in the BM upon transplantation. We found that Glypican-3 (GPC3), a heparan sulfate proteoglycan expressed on murine as well as human HSPCs, mediated this effect. TFPI did not affect CD26 activity, migration, or homing of GPC3(-/-) HSPCs, while it affected GPC1(-/-) HSPCs similar to wild-type HSPCs. Moreover, proliferation of GPC3(-/-) but not GPC1(-/-) BM HSPCs was significantly increased, which was associated with a decrease in the primitive HSC pool in BM and an increase in proportion of the circulating HSPCs in the peripheral blood. Hence, we present a novel role for TFPI and GPC3 in regulating HSC homing as well as retention in the BM.