ABSTRACT
Efficient incorporation of modified nucleotides by DNA polymerases is essential for many cutting-edge biomolecular technologies. The present study compares the acceptance of either alkene- or alkyne-modified nucleotides by KlenTaq DNA polymerase and provides structural insights into how 7-deaza-adenosine and deoxyuridine with attached alkene-modifications are incorporated into the growing DNA strand. Thereby, we identified modified nucleotides that prove to be superior substrates for KlenTaq DNA polymerase compared with their natural analogues. The knowledge can be used to guide future design of functionalized nucleotide building blocks.
Subject(s)
Alkenes/chemistry , Alkynes/chemistry , DNA-Directed DNA Polymerase/metabolism , Nucleotides/metabolism , Biocatalysis , Deoxyuridine/chemical synthesis , Deoxyuridine/chemistry , Deoxyuridine/metabolism , Electrophoresis, Polyacrylamide Gel , Nucleic Acid Amplification Techniques , Nucleotides/chemical synthesis , Nucleotides/chemistry , Tubercidin/chemical synthesis , Tubercidin/chemistry , Tubercidin/metabolismABSTRACT
Multi-valent inhibitors offer promise for the enhancement of therapeutic compounds across a range of chemical and biological processes. Here, a significant increase in enzyme-inhibition potencies was observed with a dimeric iminosugar-templated fucosidase inhibitor (IC50 = 0.108 µM) when compared to its monovalent equivalent (IC50 = 2.0 µM). Such a gain in binding is often attributed to a "multivalent effect" rising from alternative recapture of the scaffolded binding epitopes. The use of control molecules such as the meso analogue (IC50 = 0.365 µM) or the enantiomer (IC50 = 569 µM), as well as structural analysis of the fucosidase-inhibitor complex, allowed a detailed analysis of the possible mechanism of action, at the molecular level. Here, the enhanced binding affinity of the dimer over the monomer can be attributed to additional interactions in non-catalytic sites as also revealed in the 3-D structure of a bacterial fucosidase inhibitor complex.
Subject(s)
Dimerization , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , alpha-L-Fucosidase/antagonists & inhibitors , Animals , Cattle , Inhibitory Concentration 50 , Models, Molecular , Protein Conformation , StereoisomerismABSTRACT
The DNA polymerase-catalyzed incorporation of modified nucleotides is employed in many biological technologies of prime importance, such as next-generation sequencing, nucleic acid-based diagnostics, transcription analysis, and aptamer selection by systematic enrichment of ligands by exponential amplification (SELEX). Recent studies have shown that 2'-deoxynucleoside triphosphates (dNTPs) that are functionalized with modifications at the nucleobase such as dyes, affinity tags, spin and redox labels, or even oligonucleotides are substrates for DNA polymerases, even if modifications of high steric demand are used. The position at which the modification is introduced in the nucleotide has been identified as crucial for retaining substrate activity for DNA polymerases. Modifications are usually attached at the C5 position of pyrimidines and the C7 position of 7-deazapurines. Furthermore, it has been shown that the nature of the modification may impact the efficiency of incorporation of a modified nucleotide into the nascent DNA strand by a DNA polymerase. This Account places functional data obtained in studies of the incorporation of modified nucleotides by DNA polymerases in the context of recently obtained structural data. Crystal structure analysis of a Thermus aquaticus (Taq) DNA polymerase variant (namely, KlenTaq DNA polymerase) in ternary complex with primer-template DNA and several modified nucleotides provided the first structural insights into how nucleobase-modified triphosphates are tolerated. We found that bulky modifications are processed by KlenTaq DNA polymerase as a result of cavities in the protein that enable the modification to extend outside the active site. In addition, we found that the enzyme is able to adapt to different modifications in a flexible manner and adopts different amino acid side-chain conformations at the active site depending on the nature of the nucleotide modification. Different "strategies" (i.e., hydrogen bonding, cation-π interactions) enable the protein to stabilize the respective protein-substrate complex without significantly changing the overall structure of the complex. Interestingly, it was also discovered that a modified nucleotide may be more efficiently processed by KlenTaq DNA polymerase when the 3'-primer terminus is also a modified nucleotide instead of a nonmodified natural one. Indeed, the modifications of two modified nucleotides at adjacent positions can interact with each other (i.e., by π-π interactions) and thereby stabilize the enzyme-substrate complex, resulting in more efficient transformation. Several studies have indicated that archeal DNA polymerases belonging to sequence family B are better suited for the incorporation of nucleobase-modified nucleotides than enzymes from family A. However, significantly less structural data are available for family B DNA polymerases. In order to gain insights into the preference for modified substrates by members of family B, we succeeded in obtaining binary structures of 9°N and KOD DNA polymerases bound to primer-template DNA. We found that the major groove of the archeal family B DNA polymerases is better accessible than in family A DNA polymerases. This might explain the observed superiority of family B DNA polymerases in polymerizing nucleotides that bear bulky modifications located in the major groove, such as modification at C5 of pyrimidines and C7 of 7-deazapurines. Overall, this Account summarizes our recent findings providing structural insight into the mechanism by which modified nucleotides are processed by DNA polymerases. It provides guidelines for the design of modified nucleotides, thus supporting future efforts based on the acceptance of modified nucleotides by DNA polymerases.
Subject(s)
DNA-Directed DNA Polymerase/chemistry , Nucleotides/chemistry , Models, Molecular , SELEX Aptamer Technique , Substrate Specificity , Thermus/enzymologyABSTRACT
A small library of divalent fucosidase inhibitors containing pyrrolidine motifs and separated by polyamino and triazole-benzylated spacers was prepared and evaluated as α-fucosidase inhibitors. Although a weak multivalent effect was observed in polyamino derived dimers, useful structural information can be deduced about the length of the bridge, the number of nitrogen atoms present and the moieties close to the pyrrolidine. Within these investigations one of the best α-fucosidase inhibitors containing a pyrrolidine framework was obtained (18, Ki = 3.7 nM).
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , alpha-L-Fucosidase/antagonists & inhibitors , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Humans , Molecular Conformation , Pyrrolidines/chemical synthesis , Structure-Activity Relationship , alpha-L-Fucosidase/metabolismABSTRACT
The synthesis and the biological evaluation of a new ferrocenyl-iminosugar conjugate designed for fucosidase inhibitory and anticancer activity is described. The compound showed strong affinity for fucosidase from bovine kidney (Ki=23 nM) and from Bacteroides thetaiotaomicron (Ki=150 nM), displaying a 10-fold tighter binding affinity for these enzymes than the previous analogs. The interaction pattern that improves binding has been evaluated through structural analysis of the inhibitor-enzyme complex. The ferrocenyl-iminosugar exhibits significant anticancer activity on MDA-MB-231 and SK-MEL28 cell lines at 100 µM.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Ferrous Compounds/pharmacology , Imino Sugars/pharmacology , alpha-L-Fucosidase/antagonists & inhibitors , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Bacteroides/enzymology , Binding Sites/drug effects , Cattle , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Ferrous Compounds/chemistry , Humans , Imino Sugars/chemistry , Kidney/enzymology , Metallocenes , Molecular Structure , Structure-Activity Relationship , alpha-L-Fucosidase/metabolismABSTRACT
Fucosidase inhibition shows potential in numerous therapeutic contexts. Substitution of fucose-like iminosugars with hydrophobic "aglycons" yields significant improvements in potency of fucosidase inhibition. Here we have prepared three new 2-aryl-3,4-dihydroxy-5-methylpyrrolidines featuring phenyl substituents in variable orientations with respect to the iminocyclitol core and at various distances from it to explore the key binding interactions that stabilise the enzyme-inhibitor complex. The presence of a triazole linker in one structure resulted in nanomolar inhibition of the fucosidase from bovine kidney (Ki =4.8 nM), thus giving rise to one of the most potent pyrrolidine-type inhibitors of this enzyme known to date.
Subject(s)
Enzyme Inhibitors/pharmacology , Imino Sugars/chemistry , Structure-Activity Relationship , alpha-L-Fucosidase/antagonists & inhibitors , alpha-L-Fucosidase/chemistry , Animals , Cattle , Chemistry Techniques, Synthetic , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Hydrophobic and Hydrophilic Interactions , Imino Sugars/pharmacology , Kidney/enzymology , Protein Conformation , Pyrrolidines/chemistry , alpha-L-Fucosidase/metabolismABSTRACT
Enhanced metabolism of fucose through fucosidase overexpression is a signature of some cancer types, thus suggesting that fucosidase-targetted ligands could play the role of drug-delivery vectors. Herein, we describe the synthesis of a new series of pyrrolidine-ferrocene conjugates, consisting of a L-fuco-configured dihydroxypyrrolidine as the fucosidase ligand armed with a cytotoxic ferrocenylamine moeity. Three-dimensional structures of several of these fucosidase inhibitors reveal transition-state-mimicking (3)E conformations. Elaboration with the ferrocenyl moiety results in sub-micromolar inhibitors of both bovine and bacterial fucosidases, with the 3D structure of the latter revealing electron density indicative of highly mobile alkylferrocene compounds. The best compounds show a strong antiproliferative effect, with up to 100% inhibition of the proliferation of MDA-MB-231 cancer cells at 50 µM.
Subject(s)
Antineoplastic Agents/chemistry , Ferrous Compounds/chemistry , Glycoside Hydrolases/chemistry , Hydrolases/chemistry , Pyrrolidines/chemistry , alpha-L-Fucosidase/chemistry , Animals , Cattle , Cell Proliferation , Drug Delivery Systems , Enzyme Inhibitors/chemistry , Humans , Metallocenes , Molecular Conformation , Molecular Structure , alpha-L-Fucosidase/antagonists & inhibitors , alpha-L-Fucosidase/metabolismABSTRACT
We prepared a series of new iminosugar-ferrocene hybrids displaying potent inhibition of fucosidase (bovine kidney) and inactivation of MDA-MB-231 breast cancer cells proliferation at low micromolar concentrations. The synthetic route brought to light an unprecedented isomerisation of a 2-ethanalylpyrrolidine.
Subject(s)
Antineoplastic Agents/chemistry , Ferrous Compounds/chemistry , Imino Sugars/chemistry , Animals , Antineoplastic Agents/pharmacology , Cattle , Cell Line, Tumor , Cell Proliferation , Drug Screening Assays, Antitumor , Ferrous Compounds/pharmacology , Imino Sugars/pharmacology , Isomerism , Metallocenes , Pyrrolidines/chemistry , alpha-L-Fucosidase/antagonists & inhibitorsABSTRACT
A straightforward functionalization of D-pentoses is reported, which affords homoallylaminopolyols in two steps and uses ion exchange chromatography as the only purification operation. The key indium-mediated allylation is effected on unprotected glycosylamines and occurs with good to excellent syn stereoselection. Validation of the synthetic utility of the method was exemplified by a 3-step synthesis of an optically active 1,2,3,6-tetrahydropyridine from D-xylose.
